Process for producing glycoprotein composition

ABSTRACT

The present invention relates to a cell into which an RNA capable of suppressing the function of an enzyme catalyzing a reaction which converts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose is introduced; a process for producing a glycoprotein using the cell; a cell into which an RNA capable of suppressing the function of an enzyme relating to modification of a sugar chain in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in the complex type N-glycoside-linked sugar chain, and an RNA capable of suppressing the function of an enzyme relating to synthesis of an intracellular sugar nucleotide, GDP-fucose, or an RNA capable of suppressing the function of a protein relating to transport of an intracellular sugar nucleotide, GDP-fucose, to the Golgi body are introduced; a process for producing a glycoprotein composition using the cell; and the like.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a cell into which an RNA capable ofsuppressing the function of an enzyme catalyzing a reaction whichconverts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose is introduced;a process for producing a glycoprotein, which comprises using the cell;an RNA used for preparing the cell; a DNA corresponding to the RNA; anda vector comprising the DNA and its complementary DNA. Also, the presentinvention relates to a cell into which an RNA capable of suppressing thefunction of an enzyme relating to modification of a sugar chain in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in the complex type N-glycoside-linkedsugar chain, and an RNA capable of suppressing the function of an enzymeprotein relating to synthesis of an intracellular sugar nucleotide,GDP-fucose, or an RNA capable of suppressing the function of a proteinrelating to transport of an intracellular sugar nucleotide, GDP-fucose,to the Golgi body are introduced; and a process for producing aglycoprotein composition using the cell. Furthermore, the presentinvention relates to a DNA comprising a DNA corresponding to an RNAcapable of suppressing the function of an enzyme relating tomodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex type N-glycoside-linked sugar chain and its complementaryDNA, and a DNA corresponding to an RNA capable of suppressing thefunction of an enzyme protein relating to synthesis of an intracellularsugar nucleotide, GDP-fucose, or an RNA capable of suppressing thefunction of a protein relating to transport of an intracellular sugarnucleotide, GDP-fucose, to the Golgi body and its complementary DNA; avector comprising the DNA; a cell into which the vector is introduced; acell into which a vector comprising a DNA corresponding to an RNAcapable of suppressing the function of an enzyme relating tomodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex type N-glycoside-linked sugar chain and its complementaryDNA, and a vector comprising a DNA corresponding to an RNA capable ofsuppressing the function of an enzyme protein relating to synthesis ofan intracellular sugar nucleotide, GDP-fucose, or an RNA capable ofsuppressing the function of a protein relating to transport of anintracellular sugar nucleotide, GDP-fucose, to the Golgi body and itscomplementary DNA are introduced; and a process for producing aglycoprotein composition using the cell.

2. Brief Description of the Background Art

As a result of rapid development of genetic engineering or cellengineering techniques, physiologically active proteins which arepresent at a trace amount in the living body can be provided stably in alarge amount to medical sites, so that they can be applied to treatmentsof many patients. Such protein medicaments are manufactured and sold asgenetically engineered medicaments or cell culture medicaments. Theseprotein medicaments are classified into a simple protein medicament inwhich a sugar chain is not concerned with its pharmacological activityand a glycoprotein medicament in which a sugar chain plays an importantrole in its physiological activity.

Erythropoietin is exemplified as a typical example of the glycoproteinmedicament in which a sugar chain plays an important role in itspharmacological activity. Erythropoietin surely has various sugar chainstructures, and it is known that it has three complex typeN-glycoside-linked tetraantenary sugar chains in which a fucose is boundto three core structures, and one O-glycoside-linked sugar chain. Thesugar chain structures are deeply related with the in vivo physiologicalactivity of erythropoietin, and the physiological activity is notinfluenced by removing the O-glycoside-linked sugar chain [Biochemistry,31, 9872 (1992), J. Biol. Chem., 267, 7703 (1992)], but thephysiological activity is lost by removing the N-glycoside-linked sugarchains [J. Biol. Chem., 265, 12127 (1990)]. Furthermore, thepharmacological activity is influenced by addition of sialyic acid tothe N-glycoside-linked sugar chains and difference of sugar chainstructures such as a branched structure [Blood, 73, 84, (1989), Proc.Natl. Acad. Sci. U.S.A., 86, 7819 (1989), British J. Cancer, 84, 3,(2001)]. Moreover, it is shown that a protein having a sugar chainstructure in which fucose is modified generally has a shorten half-lifein blood [Science, 295, 1898 (2002)].

Regarding antibodies, it is known that the pharmacological activity isgreatly influenced by the sugar chain structures.

In the Fc region of an IgG type antibody molecule, twoN-glycoside-linked sugar chain binding sites are present. In serum IgG,a complex type sugar chain has plural branches in which sialic acid orbisecting N-acetylglucosamine are added at a low ratio is bound to thesugar chain binding site. The addition of galactose to the non-reducingend of the complex sugar chain and the addition of fucose to theN-acetylglucosamine in the reducing end is diversity [Biochemistry, 36,130 (1997)]. The sugar chain structure, that is, fucose which is addedto N-acetylglucosamine in the reducing end in the N-glycoside-linkedsugar chain which is bound to the antibody Fc region, plays an importantrole in effector functions of an antibody, such as antibody-dependentcell-mediated cytotoxic activity (hereinafter referred to as “ADCCactivity”) and complement-dependent cytotoxic activity (hereinafterreferred to as “CDC activity”) [WO00/61739, WO02/31140, J. Biol. Chem.,277, 26733 (2002), J. Biol. Chem., 278, 3466 (2003)].

Many of glycoproteins which are considered to be applied to medicamentsare produced by using recombinant DNA techniques, and manufactured byusing, as a host cell, an animal cell such as a CHO cell derived from aChinese hamster ovary tissue. However, the sugar chain structures of theglycoproteins produced by using the recombinant DNA techniques aredifferent depending on the host cells [J. Biol. Chem., 278, 3466 (2003),Glycobiology, 5, 813, (1995)]. Accordingly, sugar chains are not alwaysadded to the glycoprotein produced by the recombinant DNA techniques soas to exert suitable pharmacological activity.

Application of inhibitors of an enzyme relating to the modification of asugar chain has been attempted as a method for controlling the activityof an enzyme relating to the modification of a sugar chain in a cell andmodifying the sugar chain structure of the produced glycoprotein.However, since the inhibitors have low specificity and it is difficultto sufficiently inhibit the target enzyme, it is difficult to surelycontrol the sugar chain structure of the produced antibody.

Furthermore, modification of a sugar chain structure of a producedglycoprotein has been attempted by introducing a gene encoding an enzymerelating to the modification of a sugar chain [J. Biol. Chem., 261,13848 (1989), Science, 252, 1668 (1991)]. When an antibody is expressedby using a CHO cell into which β1,4-N-acetylglucosamine transferase III(GnTIII) is introduced, the antibody had ADCC activity 16 times higherthan the antibody expressed by using the parent cell [Glycobiology, 5,813 (1995), WO99/54342]. However, since it has been reported that excessexpression of GnTIII or β-1,4-N-acetylglucosamine transferase V (GnTV)shows toxicity for CHO cells, it is not suitable for the production oftherapeutic antibodies.

Production examples of a glycoprotein in which the produced sugar chainstructure was changed by using, as the host cell, a mutant in which theactivity of a gene encoding an enzyme relating to the modification of asugar chain was changed have been reported. The mutant in which theactivity of an enzyme relating to the modification of a sugar chain ischanged has been obtained, for example, as clones showing resistance toa lectin such as WGA (wheat-germ agglutinin derived from T. vulgaris),ConA (concanavalin A derived from C. ensiformis), RIC (a toxin derivedfrom R. communis), L-PHA (leukoagglutinin derived from P. vulgaris), LCA(lentil agglutinin derived from L. culinaris), PSA (pea lectin derivedfrom P. sativum) [Somatic Cell Mol. Genet., 12, 51 (1986)]. A case hasbeen reported in which a glycoprotein having a changed sugar chainstructure is produced by using, as the host cell, such a mutant in whichthe activity of an enzyme relating to the modification of a sugar chainwas changed. Specific examples include a report on the production of anantibody having a high mannose type sugar chain structure using a CHOcell mutant clone in which the activity of N-acetylglucosaminetransferase I (GnTI) was deleted [J. Immunol., 160, 3393 (1998)]. Inaddition, a case has been reported on the expression of an antibodyhaving a sugar chain structure in which sialic acid is not added to thenon-reducing end in the sugar chains or an antibody without addition ofgalactose thereto, using a CMP-sialic acid transporter- or UDP-galactosetransporter-deficient clone, but expression of an antibody havingimproved effector activity suitable for application to a medicament hasbeen unsuccessful [J. Immunol., 160, 3393 (1998)].

Under such a situation, it has been recently reported that an antibodyhaving high ADCC activity which is suitable for medical applications canbe produced by using, as the host cell, a clone having decreasedactivity of GDP-mannose 4,6-dehydratase (hereinafter also referred to as“GMD”), which is an enzyme catalyzing a reaction which convertsGDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose in the de novo pathwayof the intracellular sugar nucleotide, GDP-fucose [WO00/61739; J. Biol.Chem., 277, 26733 (2002); J. Biol. Chem.; 278, 3466 (2003)]. In thesereports, a clone resistant to a lectin which can recognize a sugar chainstructure in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end in the complex typeN-glycoside-linked sugar chain through α-bond, such as clone CHO-AALwhich is resistant to AAL (a lectin derived from Aleuria aurantia),clone CHO-LCA which is resistant to LCA (lentil agglutinin derived fromL. culinaris) or clone Lec 13 is used as the host cell. In addition tothese, PL^(R)1.3 established as a PSA (pea lectin derived from P.sativum)-resistant mutant of a mouse leukemia-derived clone BW 5147 isalso known as a clone having decreased activity of GDP-mannose4,6-dehydratase [J. Biol. Chem., 255, 9900 (1980)].

However, since each of these clones is not a complete gene deficientclone, it is difficult to allow an antibody to carry a sugar chainstructure which is a cause of showing high ADCC activity by theantibody, i.e. it is difficult to completely suppress an addition offucose to the N-acetylglucosamine in the reducing end in theN-glycoside-linked sugar chains. Also, since mutants such as PL^(R)1.3and Lec13 are obtained by randomly introducing mutation through amutagen treatment, they are not suitable as clones to be used in theproduction of pharmaceutical preparations.

As is described above, attempts have been made for controlling theactivity of an enzyme or protein relating to the modification of a sugarchain in a host cell in order to modify the sugar chain structure of aproduced glycoprotein. However, since the modification mechanism of thesugar chain is various and complicated and the physiological functionsof the sugar chain have not been sufficiently solved, trial and errorare repeated at present. Especially, although a clone in which theactivity of an enzyme catalyzing a reaction which converts GDP-mannoseinto GDP-4-keto,6-deoxy-GDP-mannose has been obtained and an antibodycomposition having high effector activity has been produced, theactivity cannot be sufficiently controlled.

As an example of attempts for simply controlling the activity of anenzyme or protein relating to the modification of a sugar chain in ahost cell, a method for controlling the function of a specific geneusing siRNA (small interfering RNA) is known (WO03/85118). Also, it isreported that a method for designing an RNA molecule used forsuppressing the function of a gene [Nature Biotech., 22, 326 (2004)].However, the RNA molecule designed by such a method is not always amolecule which can efficiently suppress the function of a target gene(Current Opinion in Molecular Therapeutics, 6, 129 (2004), and thedesign of an RNA molecule showing effective functional suppressiveeffect on a specific gene involves trial and error.

Also, it is shown that modification of binding of a fucose to a sugarchain which is added to a produced glycoprotein can be controlled byusing a cell into which an RNA capable of suppressing the function of anenzyme relating to modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain isintroduced (WO02/31140, WO03/85118). These reports show that the ratioof a sugar chain in which fucose is not bound among sugar chains boundto a produced antibody molecule can be increased by introducing an RNAcapable of suppressing the function of α1,6-fucosyltransferase into acell line which produces an antibody molecule.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a cell into which anRNA capable of suppressing the function of an enzyme catalyzing areaction which converts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannoseis introduced; a process for producing a glycoprotein composition usingthe cell; an RNA used for preparing the cell; a DNA corresponding to theRNA; and a vector comprising the DNA and its complementary DNA.

Also, an object of the present invention is to provide a cell into whichan RNA capable of suppressing the function of an enzyme relating tomodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex type N-glycoside-linked sugar chain, and an RNA capable ofsuppressing the function of an enzyme protein relating to synthesis ofan intracellular sugar nucleotide, GDP-fucose, or an RNA capable ofsuppressing the function of a protein relating to transport of anintracellular sugar nucleotide, GDP-fucose, to the Golgi body areintroduced; and a process for producing a glycoprotein composition usingthe cell.

Furthermore, an object of the present invention is to provide a DNAcomprising a DNA corresponding to an RNA capable of suppressing thefunction of an enzyme relating to modification of a sugar chain in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in the complex type N-glycoside-linkedsugar chain and its complementary DNA, and a DNA corresponding to an RNAcapable of suppressing the function of an enzyme protein relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, or an RNAcapable of suppressing the function of a protein relating to transportof an intracellular sugar nucleotide, GDP-fucose, to the Golgi body andits complementary DNA; a vector comprising the DNA; a cell into whichthe vector is introduced; a cell into which a vector comprising a DNAcorresponding to an RNA capable of suppressing the function of an enzymerelating to modification of a sugar chain in which 1-position of fucoseis bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain andits complementary DNA, and a vector comprising a DNA corresponding to anRNA capable of suppressing the function of an enzyme protein relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, or an RNAcapable of suppressing the function of a protein relating to transportof an intracellular sugar nucleotide, GDP-fucose, to the Golgi body andits complementary DNA are introduced; and a process for producing aglycoprotein composition using the cell.

The present invention relates to the following (1) to (71):

(1) A cell into which a double-stranded RNA comprising an RNA selectedfrom the following (a) or (b) and its complementary RNA are introduced:

(a) an RNA comprising the nucleotide sequence represented by SEQ IDNO:37, 57 or 58;

(b) an RNA consisting of a nucleotide sequence in which one or a severalnucleotide(s) is/are deleted, substituted, inserted and/or added in thenucleotide sequence represented by SEQ ID NO:37, 57 or 58 and havingactivity of suppressing the function of an enzyme catalyzing a reactionwhich converts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose.

(2) The cell according to (1), wherein the enzyme catalyzing adehydration reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose is GDP-mannose 4,6-dehydratase.

(3) The cell according to (2), wherein the GDP-mannose 4,6-dehydrataseis a protein encoded by a DNA selected from the group consisting of thefollowing (a) to (f):

(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:8;

(b) a DNA comprising the nucleotide sequence represented by SEQ ID NO:9;

(c) a DNA comprising the nucleotide sequence represented by SEQ IDNO:10;

(d) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:8 under stringent conditions andencodes a protein having GDP-mannose 4,6-dehydratase activity;

(e) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:9 under stringent conditions andencodes a protein having GDP-mannose 4,6-dehydratase activity;

(f) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:10 under stringent conditions andencodes a protein having GDP-mannose 4,6-dehydratase activity.

(4) The cell according to (2), wherein the GDP-mannose 4,6-dehydrataseis a protein selected from the group consisting of the following (a) to(i):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:11;

(b) a protein comprising the amino acid sequence represented by SEQ IDNO:12;

(c) a protein comprising the amino acid sequence represented by SEQ IDNO:13;

(d) a protein consisting of an amino acid sequence in which one or aseveral amino acid(s) is/are deleted, substituted, inserted and/or addedin the amino acid sequence represented by SEQ ID NO:11 and havingGDP-mannose 4,6-dehydratase activity;

(e) a protein consisting of an amino acid sequence in which one or aseveral amino acid(s) is/are deleted, substituted, inserted and/or addedin the amino acid sequence represented by SEQ ID NO:12 and havingGDP-mannose 4,6-dehydratase activity;

(f) a protein consisting of an amino acid sequence in which one or aseveral amino acid(s) is/are deleted, substituted, inserted and/or addedin the amino acid sequence represented by SEQ ID NO:13 and havingGDP-mannose 4,6-dehydratase activity;

(g) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:11 andhaving GDP-mannose 4,6-dehydratase activity;

(h) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:12 andhaving GDP-mannose 4,6-dehydratase activity;

(i) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:13 andhaving GDP-mannose 4,6-dehydratase activity.

(5) A double-stranded RNA comprising an RNA selected from the following(a) or (b) and its complementary RNA:

(a) an RNA comprising the nucleotide sequence represented by SEQ IDNO:37, 57 or 58;

(b) an RNA consisting of a nucleotide sequence in which one or a severalnucleotide(s) is/are deleted, substituted, inserted and/or added in thenucleotide sequence represented by SEQ ID NO:37, 57 or 58 and havingactivity of suppressing the function of an enzyme catalyzing a reactionwhich converts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose.

(6) A DNA corresponding to the RNA according to (5) and itscomplementary DNA.

(7) A vector comprising a DNA corresponding to the RNA according to (5).

(8) A cell into which the vector according to (7) is introduced.

(9) A cell into which an RNA capable of suppressing the function of anenzyme relating to modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain, andan RNA capable of suppressing the function of an enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, or an RNAcapable of suppressing the function of a protein relating to transportof an intracellular sugar nucleotide, GDP-fucose, to the Golgi body areintroduced.

(10) A cell into which an RNA capable of suppressing the function of anenzyme relating to modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain, andan RNA capable of suppressing the function of an enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, areintroduced.

(11) The cell according to (9) or (10), wherein the enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, is an enzymecatalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose.

(12) The cell according to any one of (9) to (11), wherein the enzymerelating to modification of a sugar chain in which 1-position of fucoseis bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain isα1,6-fucosyltransferase.

(13) The cell according to (12), wherein the α1,6-fucosyltransferase isa protein encoded by a DNA selected from the group consisting of (a) to(h):

(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1;

(b) a DNA comprising the nucleotide sequence represented by SEQ ID NO:2;

(c) a DNA comprising the nucleotide sequence represented by SEQ ID NO:3;

(d) a DNA comprising the nucleotide sequence represented by SEQ ID NO:4;

(e) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:1 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity;

(f) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:2 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity;

(g) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:3 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity;

(h) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:4 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity.

(14) The cell according to (12), wherein the α1,6-fucosyltransferase isa protein selected from the group consisting of the following (a) to(l):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:5;

(b) a protein comprising the amino acid sequence represented by SEQ IDNO:6;

(c) a protein comprising the amino acid sequence represented by SEQ IDNO:7;

(d) a protein comprising the amino acid sequence represented by SEQ IDNO:84;

(e) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:5 and havingα1,6-fucosyltransferase activity;

(f) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:6 and havingα1,6-fucosyltransferase activity;

(g) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:7 and havingα1,6-fucosyltransferase activity;

(h) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:84 and havingα1,6-fucosyltransferase activity;

(i) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:5 andhaving α1,6-fucosyltransferase activity;

(j) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:6 andhaving α1,6-fucosyltransferase activity;

(k) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:7 andhaving α1,6-fucosyltransferase activity;

(l) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:84 andhaving α1,6-fucosyltransferase activity.

(15) The cell according to any one of (9) to (14), wherein the RNAcapable of suppressing the function of an enzyme relating tomodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex type N-glycoside-linked sugar chain is a double-stranded RNAcomprising an RNA selected from the group consisting of the following(a) to (d) and its complementary RNA:

(a) an RNA corresponding to a DNA comprising a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:1;

(b) an RNA corresponding to a DNA comprising a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:2;

(c) an RNA corresponding to a DNA comprising a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:3;

(d) an RNA corresponding to a DNA comprising a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:4.

(16) The cell according to any one of (9) to (14), wherein the RNAcapable of suppressing the function of an enzyme relating tomodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex type N-glycoside-linked sugar chain is a double-stranded RNAcomprising an RNA selected from the group consisting of the following(a) and (b) and its complementary RNA:

(a) an RNA comprising the nucleotide sequence represented by any one ofSEQ ID NO:14 to 35 or 85 to 89;

(b) an RNA consisting of a nucleotide sequence in which one or a severalnucleotide(s) is/are deleted, substituted, inserted and/or added in thenucleotide sequence represented by any one of SEQ ID NO:14 to 35 or 85to 89 and having activity of suppressing the function ofα1,6-fucosyltransferase activity.

(17) The cell according to any one of (11) to (16), wherein the enzymecatalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose is GDP-mannose 4,6-dehydratase.

(18) The cell according to (17), wherein the GDP-mannose 4,6-dehydrataseis a protein encoded by a DNA selected from the group consisting of thefollowing (a) to (f):

(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:8;

(b) a DNA comprising the nucleotide sequence represented by SEQ ID NO:9;

(c) a DNA comprising the nucleotide sequence represented by SEQ IDNO:10;

(d) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:8 under stringent conditions andencodes a protein having GDP-mannose 4,6-dehydratase activity;

(e) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:9 under stringent conditions andencodes a protein having GDP-mannose 4,6-dehydratase activity;

(f) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:10 under stringent conditions andencodes a protein having GDP-mannose 4,6-dehydratase activity.

(19) The cell according to (17), wherein the GDP-mannose 4,6-dehydrataseis a protein selected from the group consisting of the following (a) to(i):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:11;

(b) a protein comprising the amino acid sequence represented by SEQ IDNO:12;

(c) a protein comprising the amino acid sequence represented by SEQ IDNO:13;

(d) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:11 and having GDP-mannose4,6-dehydratase activity;

(e) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:12 and having GDP-mannose4,6-dehydratase activity;

(f) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:13 and having GDP-mannose4,6-dehydratase activity;

(g) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:11 andhaving GDP-mannose 4,6-dehydratase activity;

(h) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:12 andhaving GDP-mannose 4,6-dehydratase activity;

(i) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:13 andhaving GDP-mannose 4,6-dehydratase activity.

(20) The cell according to any one of (11) to (19), wherein the RNAcapable of suppressing the function of an enzyme catalyzing a reactionwhich converts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose is adouble-stranded RNA comprising an RNA selected from the group consistingof the following (a) to (c) and its complementary RNA:

(a) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:8;

(b) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:9;

(c) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:10.

(21) The cell according to any one of (11) to (19), wherein the RNAcapable of suppressing the function of an enzyme catalyzing a reactionwhich converts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose is adouble-stranded RNA comprising an RNA selected from the group consistingof the following (a) and (b) and its complementary RNA:

(a) an RNA comprising the nucleotide sequence represented by any one ofSEQ ID NO:37, 57 or 58;

(b) an RNA consisting of a nucleotide sequence in which one or a severalnucleotide(s) is/are deleted, substituted, inserted and/or added in thenucleotide sequence represented by any one of SEQ ID NO:37, 57 or 58 andhaving activity of suppressing the function of GDP-mannose4,6-dehydratase.

(22) A DNA comprising a DNA corresponding to an RNA capable ofsuppressing the function of an enzyme relating to modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complextype N-glycoside-linked sugar chain and its complementary DNA, and a DNAcorresponding to an RNA capable of suppressing the function of an enzymerelating to synthesis of an intracellular sugar nucleotide, GDP-fucose,and its complementary DNA or a DNA corresponding to an RNA capable ofsuppressing the function of a protein relating to transport of anintracellular sugar nucleotide, GDP-fucose, to the Golgi body and itscomplementary DNA.

(23) A DNA comprising a DNA corresponding to an RNA capable ofsuppressing the function of an enzyme relating to modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complextype N-glycoside-linked sugar chain and its complementary DNA, and a DNAcorresponding to an RNA capable of suppressing the function of an enzymerelating to synthesis of an intracellular sugar nucleotide, GDP-fucose,and its complementary DNA.

(24) The DNA according to (22) or (23), wherein the enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, is an enzymecatalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose.

(25) The DNA according to any one of (22) to (24), wherein the enzymerelating to modification of a sugar chain in which 1-position of fucoseis bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain isα1,6-fucosyltransferase.

(26) The DNA according to (25), wherein the α1,6-fucosyltransferase is aprotein encoded by a DNA selected from the group consisting of (a) to(h):

(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1;

(b) a DNA comprising the nucleotide sequence represented by SEQ ID NO:2;

(c) a DNA comprising the nucleotide sequence represented by SEQ ID NO:3;

(d) a DNA comprising the nucleotide sequence represented by SEQ ID NO:4;

(e) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:1 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity;

(f) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:2 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity;

(g) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:3 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity;

(h) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:4 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity.

(27) The DNA according to (25), wherein the α1,6-fucosyltransferase is aprotein selected from the group consisting of the following (a) to (l):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:5;

(b) a protein comprising the amino acid sequence represented by SEQ IDNO:6;

(c) a protein comprising the amino acid sequence represented by SEQ IDNO:7;

(d) a protein comprising the amino acid sequence represented by SEQ IDNO:84;

(e) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:5 and havingα1,6-fucosyltransferase activity;

(f) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:6 and havingα1,6-fucosyltransferase activity;

(g) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:7 and havingα1,6-fucosyltransferase activity;

(h) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:84 and havingα1,6-fucosyltransferase activity;

(i) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:5 andhaving α1,6-fucosyltransferase activity;

(j) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:6 andhaving α1,6-fucosyltransferase activity;

(k) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:7 andhaving α1,6-fucosyltransferase activity;

(l) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:84 andhaving α1,6-fucosyltransferase activity.

(28) The DNA according to any one of (22) to (27), wherein the RNAcapable of suppressing the function of an enzyme relating tomodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex type N-glycoside-linked sugar chain is an RNA selected fromthe group consisting of the following (a) to (d):

(a) an RNA corresponding to a DNA comprising a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:1;

(b) an RNA corresponding to a DNA comprising a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:2;

(c) an RNA corresponding to a DNA comprising a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:3;

(d) an RNA corresponding to a DNA comprising a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:4.

(29) The DNA according to any one of (22) to (27), wherein the RNAcapable of suppressing the function of an enzyme relating tomodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex type N-glycoside-linked sugar chain is an RNA selected fromthe group consisting of the following (a) and (b):

(a) an RNA comprising the nucleotide sequence represented by any one ofSEQ ID NO:14 to 35 or 85 to 89;

(b) an RNA consisting of a nucleotide sequence in which one or a severalnucleotide(s) is/are deleted, substituted, inserted and/or added in thenucleotide sequence represented by any one of SEQ ID NO:14 to 35 or 85to 89 and having activity of suppressing the function ofα1,6-fucosyltransferase activity.

(30) The DNA according to any one of (24) to (29), wherein the enzymecatalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose is GDP-mannose 4,6-dehydratase.

(31) The DNA according to (30), wherein the GDP-mannose 4,6-dehydrataseis a protein encoded by a DNA selected from the group consisting of thefollowing (a) to (f):

(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:8;

(b) a DNA comprising the nucleotide sequence represented by SEQ ID NO:9;

(c) a DNA comprising the nucleotide sequence represented by SEQ IDNO:10;

(d) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:8 under stringent conditions andencodes a protein having GDP-mannose 4,6-dehydratase activity;

(e) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:9 under stringent conditions andencodes a protein having GDP-mannose 4,6-dehydratase activity;

(f) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:10 under stringent conditions andencodes a protein having GDP-mannose 4,6-dehydratase activity.

(32) The DNA according to (30), wherein the GDP-mannose 4,6-dehydrataseis a protein selected from the group consisting of the following (a) to(i):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:11;

(b) a protein comprising the amino acid sequence represented by SEQ IDNO:12;

(c) a protein comprising the amino acid sequence represented by SEQ IDNO:13;

(d) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:11 and having GDP-mannose4,6-dehydratase activity;

(e) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:12 and having GDP-mannose4,6-dehydratase activity;

(f) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:13 and having GDP-mannose4,6-dehydratase activity;

(g) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:11 andhaving GDP-mannose 4,6-dehydratase activity;

(h) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:12 andhaving GDP-mannose 4,6-dehydratase activity;

(i) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:13 andhaving GDP-mannose 4,6-dehydratase activity.

(33) The DNA according to any one of (24) to (32), wherein the RNAcapable of suppressing the function of an enzyme catalyzing a reactionwhich converts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose is an RNAselected from the group consisting of the following (a) to (c):

(a) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:8;

(b) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:9;

(c) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:10.

(34) The DNA according to any one of (24) to (32), wherein the RNAcapable of suppressing the function of an enzyme catalyzing a reactionwhich converts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose is an RNAselected from the group consisting of the following (a) and (b):

(a) an RNA comprising the nucleotide sequence represented by any one ofSEQ ID NO:37, 57 or 58;

(b) an RNA consisting of a nucleotide sequence in which one or a severalnucleotide(s) is/are deleted, substituted, inserted and/or added in thenucleotide sequence represented by any one of SEQ ID NO:37, 57 or 58 andhaving activity of suppressing the function of GDP-mannose4,6-dehydratase.

(35) A vector comprising the DNA according to any one of (22) to (34).

(36) The vector according to (35), which comprises the DNA representedby SEQ ID NO:90 and the DNA represented by SEQ ID NO:92.

(37) The vector according to (35), which comprises the DNA representedby SEQ ID NO:91 and the DNA represented by SEQ ID NO:92.

(38) The vector according to (35), which comprises the DNA representedby SEQ ID NO:90 and the DNA represented by SEQ ID NO:93.

(39) The vector according to (35), which comprises the DNA representedby SEQ ID NO:91 and the DNA represented by SEQ ID NO:93.

(40) A cell into which the vector according to any one of (35) to (39)is introduced.

(41) A cell into which a vector comprising a DNA corresponding to an RNAcapable of suppressing the function of an enzyme relating tomodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex type N-glycoside-linked sugar chain and its complementaryDNA, and a vector comprising a DNA corresponding to an RNA capable ofsuppressing the function of an enzyme relating to synthesis of anintracellular sugar nucleotide, GDP-fucose, and its complementary DNA ora vector comprising a DNA corresponding to an RNA capable of suppressingthe function of a protein relating to transport of an intracellularsugar nucleotide, GDP-fucose, to the Golgi body and its complementaryDNA are introduced.

(42) A cell into which a vector comprising a DNA corresponding to an RNAcapable of suppressing the function of an enzyme relating tomodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex type N-glycoside-linked sugar chain and its complementaryDNA, and a vector comprising a DNA corresponding to an RNA capable ofsuppressing the function of an enzyme relating to synthesis of anintracellular sugar nucleotide, GDP-fucose, and its complementary DNAare introduced.

(43) The cell according to (41) or (42), wherein the RNA capable ofsuppressing the function of an enzyme protein relating to synthesis ofan intracellular sugar nucleotide, GDP-fucose, is an RNA capable ofsuppressing the function of an enzyme catalyzing a reaction whichconverts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose.

(44) The cell according to any one of (41) to (43), wherein the enzymerelating to modification of a sugar chain in which 1-position of fucoseis bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain isα1,6-fucosyltransferase.

(45) The cell according to (44), wherein the α1,6-fucosyltransferase isa protein encoded by a DNA selected from the group consisting of (a) to(h):

(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1;

(b) a DNA comprising the nucleotide sequence represented by SEQ ID NO:2;

(c) a DNA comprising the nucleotide sequence represented by SEQ ID NO:3;

(d) a DNA comprising the nucleotide sequence represented by SEQ ID NO:4;

(e) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:1 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity;

(f) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:2 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity;

(g) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:3 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity;

(h) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:4 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity.

(46) The cell according to (44), wherein the α1,6-fucosyltransferase isa protein selected from the group consisting of the following (a) to(l):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:5;

(b) a protein comprising the amino acid sequence represented by SEQ IDNO:6;

(c) a protein comprising the amino acid sequence represented by SEQ IDNO:7;

(d) a protein comprising the amino acid sequence represented by SEQ IDNO:84;

(e) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:5 and havingα1,6-fucosyltransferase activity;

(f) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:6 and havingα1,6-fucosyltransferase activity;

(g) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:7 and havingα1,6-fucosyltransferase activity;

(h) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:84 and havingα1,6-fucosyltransferase activity;

(i) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:5 andhaving α1,6-fucosyltransferase activity;

(j) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:6 andhaving α1,6-fucosyltransferase activity;

(k) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:7 andhaving α1,6-fucosyltransferase activity;

(l) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:84 andhaving α1,6-fucosyltransferase activity.

(47) The cell according to any one of (41) to (46), wherein the RNAcapable of suppressing the function of an enzyme relating tomodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex type N-glycoside-linked sugar chain is a double-stranded RNAcomprising an RNA selected from the group consisting of the following(a) to (d) and its complementary RNA:

(a) an RNA corresponding to a DNA comprising a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:1;

(b) an RNA corresponding to a DNA comprising a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:2;

(c) an RNA corresponding to a DNA comprising a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:3;

(d) an RNA corresponding to a DNA comprising a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:4.

(48) The cell according to any one of (41) to (46), wherein the RNAcapable of suppressing the function of an enzyme relating tomodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex type N-glycoside-linked sugar chain is a double-stranded RNAcomprising an RNA selected from the group consisting of the following(a) and (b) and its complementary RNA:

(a) an RNA comprising the nucleotide sequence represented by any one ofSEQ ID NO:14 to 35 or 85 to 89;

(b) an RNA consisting of a nucleotide sequence in which one or a severalnucleotide(s) is/are deleted, substituted, inserted and/or added in thenucleotide sequence represented by any one of SEQ ID NO:14 to 35 or 85to 89 and having activity of suppressing the function ofα1,6-fucosyltransferase activity.

(49) The cell according to any one of (43) to (48), wherein the enzymecatalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose is GDP-mannose 4,6-dehydratase.

(50) The cell according to (49), wherein the GDP-mannose 4,6-dehydrataseis a protein encoded by a DNA selected from the group consisting of thefollowing (a) to (f):

(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:8;

(b) a DNA comprising the nucleotide sequence represented by SEQ ID NO:9;

(c) a DNA comprising the nucleotide sequence represented by SEQ IDNO:10;

(d) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:8 under stringent conditions andencodes a protein having GDP-mannose 4,6-dehydratase activity;

(e) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:9 under stringent conditions andencodes a protein having GDP-mannose 4,6-dehydratase activity;

(f) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:10 under stringent conditions andencodes a protein having GDP-mannose 4,6-dehydratase activity.

(51) The cell according to (49), wherein the GDP-mannose 4,6-dehydrataseis a protein selected from the group consisting of the following (a) to(i):

(a) a protein comprising the amino acid sequence represented by SEQ IDNO:11;

(b) a protein comprising the amino acid sequence represented by SEQ IDNO:12;

(c) a protein comprising the amino acid sequence represented by SEQ IDNO:13;

(d) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:11 and having GDP-mannose4,6-dehydratase activity;

(e) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:12 and having GDP-mannose4,6-dehydratase activity;

(f) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:13 and having GDP-mannose4,6-dehydratase activity;

(g) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:11 andhaving GDP-mannose 4,6-dehydratase activity;

(h) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:12 andhaving GDP-mannose 4,6-dehydratase activity;

(i) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:13 andhaving GDP-mannose 4,6-dehydratase activity.

(52) The cell according to any one of (43) to (51), wherein the RNAcapable of suppressing the function of an enzyme catalyzing a reactionwhich converts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose is adouble-stranded RNA comprising an RNA selected from the group consistingof the following (a) to (c) and its complementary RNA:

(a) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:8;

(b) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:9;

(c) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:10.

(53) The cell according to any one of (43) to (51), wherein the RNAcapable of suppressing the function of an enzyme catalyzing a reactionwhich converts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose is adouble-stranded RNA comprising an RNA selected from the group consistingof the following (a) and (b) and its complementary RNA:

(a) an RNA comprising the nucleotide sequence represented by any one ofSEQ ID NO:37, 57 or 58;

(b) an RNA consisting of a nucleotide sequence in which one or a severalnucleotide(s) is/are deleted, substituted, inserted and/or added in thenucleotide sequence represented by any one of SEQ ID NO:37, 57 or 58 andhaving activity of suppressing the function of GDP-mannose4,6-dehydratase.

(54) The cell according to any one of (1) to (4), (8) to (21) and (40)to (53), which is resistant to a lectin which recognizes a sugar chainstructure in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in anN-glycoside-linked sugar chain.

(55) The cell according to (54), wherein the lectin is selected from thegroup consisting of the following (a) to (d):

(a) a Lens culinaris agglutinin LCA (lentil agglutinin derived from Lensculinaris);

(b) a Pisum sativum agglutinin PSA (pea lectin derived from Pisumsativum);

(c) a Vicia faba agglutinin VFA (agglutinin derived from Vicia faba);

(d) an Aleuria aurantia lectin AAL (lectin derived from Aleuriaaurantia).

(56) The cell according to any one of (1) to (4), (8) to (21) and (40)to (55), which is a cell selected from the group consisting of a yeast,an animal cell, an insect cell and a plant cell.

(57) The cell according to (56), wherein the animal cell is selectedfrom the group consisting of the following (a) to (k):

(a) a CHO cell derived from a Chinese hamster ovary tissue;

(b) a rat myeloma cell line YB2/3HL.P2.G11.16Ag.20 cell;

(c) a mouse myeloma cell line NS0 cell;

(d) a mouse myeloma cell line SP2/0-Ag14 cell;

(e) a BHK cell derived from a Syrian hamster kidney tissue;

(f) a hybridoma cell which produces an antibody;

(g) a human leukemic cell line Namalwa cell;

(h) a human leukemic cell line NM-F9 cell;

(i) a human embryonic retinal cell line PER.C6 cell;

(j) an embryonic stem cell;

(k) a fertilized egg cell.

(58) The cell according to any one of (1) to (4), (8) to (21) and (40)to (57), which comprises a gene encoding a glycoprotein.

(59) The cell according to (58), wherein the glycoprotein is an antibodymolecule.

(60) The cell according to (59), wherein the antibody molecule isselected from the group consisting of the following (a) to (d):

(a) a human antibody;

(b) a humanized antibody;

(c) an antibody fragment comprising the Fc region of (a) or (b);

(d) a fusion protein comprising the Fc region of (a) or (b).

(61) The cell according to (59) or (60), wherein the antibody moleculebelongs to an IgG class.

(62) A process for producing a glycoprotein composition, which comprisesusing the cell according to (58).

(63) A process for producing a glycoprotein composition, which comprisesculturing the cell according to (58) in a medium to form and accumulatethe glycoprotein composition in the culture; and recovering andpurifying the glycoprotein composition from the culture.

(64) A process for producing an antibody composition, which comprisesusing the cell according to any one of (59) to (61).

(65) A process for producing an antibody composition, which comprisesculturing the cell according to any one of (59) to (61) in a medium toform and accumulate the antibody composition in the culture; andrecovering and purifying the antibody composition from the culture.

(66) The process according to (64) or (65), wherein the antibodycomposition is an antibody composition having a higherantibody-dependent cell-mediated cytotoxic activity than an antibodycomposition produced by its parent cell.

(67) The process according to (66), wherein the antibody compositionhaving a higher antibody-dependent cell-mediated cytotoxic activity hasa higher ratio of a sugar chain in which fucose is not bound toN-acetylglucosamine in the reducing end in the sugar chain among totalcomplex type N-glycoside-linked sugar chains bound to the Fc region inthe antibody composition than an antibody composition produced by itsparent cell.

(68) The process according to (67), wherein the sugar chain in whichfucose is not bound is a sugar chain in which 1-position of the fucoseis not bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in a complex type N-glycoside-linked sugar chain.

(69) A glycoprotein composition produced by the process according to(62) or (63).

(70) An antibody composition produced by the process according to anyone of (64) to (68).

(71) A medicament comprising the composition according to (69) or (70)as an active ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the construction of plasmid pBS-U6term.

FIG. 2 shows the construction of plasmid pPUR-U6term.

FIG. 3 shows the construction of plasmid pPUR/GMDshB.

FIG. 4 shows the amount of GMD mRNA in each clone. The abscissaindicates the relative amount of GMD mRNA to the amount of β-actin mRNAwhen the amount of the parent clone 32-05-12 was assumed to be 100, andthe ordinate indicates each clones. In the drawing, the black barindicates the parent clone into which siRNA was not introduced, and theoutline bars indicate the clones into which the GMD-targeting siRNAexpression vector was introduced alone.

FIG. 5 shows changes of viable cell densities of clone 32-05-12AF whichwas CHO/DG44 cell-derived anti-CCR4 antibody-producing clone, andlectin-resistant clones, 12-GMDB-2AF and 12-GMDB-5AF, into which theGMD-targeting siRNA expression plasmid was introduced through serum-freefed-batch culture. The abscissa and the ordinate indicate the culturingdays and the viable cell density, respectively. In the drawing, closedtriangles, open circles and closed circles indicate clone 32-05-12AF,clone 12-GMDB-2AF and clone 12-GMDB-5, respectively.

FIG. 6 shows changes of the amount of antibody accumulated in culturesupernatant of serum-free fed-batch culture of clone 32-05-12AF whichwas CHO/DG44 cell-derived anti-CCR4 antibody-producing clone, andlectin-resistant clones, 12-GMDB-2AF and 12-GMDB-5AF, introduced withthe GMD-targeting siRNA expression plasmid. The abscissa and theordinate indicate the culturing days and the amount of antibodyaccumulated, respectively. In the drawing, closed triangles, opencircles and closed circles indicate clone 32-05-12AF, clone 12-GMDB-2AFand clone 12-GMDB-5, respectively.

FIG. 7 shows the constructions of plasmids FUT8shRNA/lib2B/pPUR andFUT8shRNA/lib3/pPUR.

FIG. 8 shows the constructions of plasmids FT8libB/pBS and FT8lib3/pBS.

FIG. 9 shows the constructions of plasmids Fr8libB/pAGE andFT8lib3/pAGE.

FIG. 10 shows the amount of GMD mRNA in each clone introduced with theGMD-targeting siRNA expression vector and the FUT8-targeting siRNAexpression vector or with the GMD-targeting siRNA expression vectoralone, and the parent clone. The abscissa indicates the relative amountof GMD mRNA to the amount of β-actin mRNA when the amount of the parentclone 32-05-12 was assumed to be 100, and the ordinate indicates eachclones. In the drawing, black and outline bars indicate the parent clonewithout siRNA introduction and clones introduced with GMD-targetingsiRNA expression vector alone, respectively.

FIG. 11 shows the amount of FUT8 mRNA in each clone introduced with theGMD-targeting siRNA expression vector and the FUT8-targeting siRNAexpression vector or with the GMD-targeting siRNA expression vectoralone, and the parent clone. The abscissa indicates the relative amountof FUT8 mRNA to the amount of β-actin mRNA when the amount in the parentclone 32-05-12 was assumed to be 100, and the ordinate indicates eachclones. In the drawing, black and outline bars indicate parent clonewithout siRNA introduction and clones introduced with the GMD-targetingsiRNA expression vector alone, respectively.

FIG. 12 shows changes of viable cell densities of clone 32-05-12AF whichwas CHO/DG44 cell-derived anti-CCR4 antibody-producing clone andlectin-resistant Wi23-5AF clone introduced with the GMD-targeting siRNAexpression vector and the FUT8-targeting siRNA expression vector inserum-free fed-batch culture. The abscissa and the ordinate indicateculturing days and viable cell density, respectively. In the drawing,dotted and solid lines indicate 32-05-12AF and Wi23-5AF clones,respectively.

FIG. 13 shows changes of the amount of antibody accumulated in culturesupernatant of serum-free fed-batch culture of clone 32-05-12AF whichwas CHO/DG44 cell-derived anti-CCR4 antibody-producing clone andlectin-resistant Wi23-5AF clone introduced with the GMD-targeting siRNAexpression vector and the FUT8-targeting siRNA expression vector. Theabscissa and the ordinate indicate culturing days and the amount ofantibody accumulated, respectively. In the drawing, dotted and solidlines indicate 32-05-12AF and Wi23-5AF clones, respectively.

FIG. 14 shows shFcγRIIIa binding activity of a standard sample in whichfucose(−)% of an anti-CCR4 chimeric antibody was known. The abscissa andthe ordinate indicate fucose(−)% and absorbance at 490 nm showingshFcγRIIIa binding activity, respectively.

FIG. 15 shows fucose(−)% calculated from the shFcγRIIIa binding activityof anti-CCR4 chimeric antibody contained in culture supernatants inserum-free fed-batch medium of clone 32-05-12AF which was CHO/DG44cell-derived anti-CCR4 antibody-producing clone or lectin-resistantWi23-5AF clone introduced with the GMD-targeting siRNA expression vectorand the FUT8-targeting siRNA expression vector. The abscissa and theordinate indicate culturing days and fucose(−)%, respectively. In thedrawing, dotted and solid lines indicate 32-05-12AF and Wi23-5AF clones,respectively.

FIG. 16 shows the constructions of plasmids pCR/GMDshB and pCR/GMDmB.

FIG. 17 shows the constructions of plasmids FT8libB_GMDB/pAGE,FT8lib3_GMDB/pAGE, FT8libB_GMDmB/pAGE and FT8lib3_GMDmB/pAGE.

FIG. 18 shows the relative amount of GMD mRNA expressed in each cloneobtained by introducing the GMD-targeting siRNA and FUT8-targeting siRNAco-expression vector into clone 32-05-12 which was CHO/DG44 cell-derivedanti-CCR4 antibody-producing clone. The abscissa indicates the relativeamount of GMD mRNA to the amount of β-actin mRNA when the amount in theparent clone 32-05-12 was assumed to be 100, and the ordinate indicateseach clones. In the drawing, black and outline bars indicate therelative amount of GMD mRNA of the parent clone and of each clonesobtained by introducing the GMD- and FUT8-targeting siRNA co-expressionvector, respectively, when the amount in the parent clone 32-05-12 wasassumed to be 100.

FIG. 19 shows the relative amount of FUT8 mRNA expressed in each cloneobtained by introducing the GMD-targeting siRNA and FUT8-targeting siRNAco-expression vector into clone 32-05-12 which was CHO/DG44 cell-derivedanti-CCR4 antibody-producing clone. The abscissa indicates the relativeamount of FUT8 mRNA to the amount of β-actin mRNA when the amount in theparent clone 32-05-12 was assumed to be 100, and the ordinate indicateseach clones. In the drawing, black and outline bars indicate therelative amount of FUT8 mRNA of the parent clone and of each clonesobtained by introducing the GMD-targeting siRNA and FUT8-targeting siRNAco-expression vector, respectively, when the amount in the parent clonewas assumed to be 100.

FIG. 20 shows the construction of plasmid pPUR/GMDmB.

FIG. 21 shows the relative amount of GMD mRNA expressed in each cloneobtained by introducing the mouse GMD-targeting siRNA and FUT8-targetingsiRNA co-expression vector into clone KM968 which was SP2/0 cell-derivedanti-GM₂ antibody-producing clone. The abscissa indicates the relativeamount of GMD mRNA to the amount of β-actin mRNA when the amount in theparent clone KM968 was assumed to be 100, and the ordinate indicateseach clones. In the drawing, black and outline bars indicate therelative amount of GMD mRNA of the parent clone and of each clonesobtained by introducing the GMD-targeting siRNA and FUT8-targeting siRNAco-expression vector, respectively, when the amount in the parent cloneKM968 was assumed to be 100.

FIG. 22 shows the relative amount of FUT8 mRNA expressed in each cloneobtained by introducing the mouse GMD-targeting siRNA and FUT8-targetingsiRNA co-expression vector into clone KM968 which was SP2/0 cell-derivedanti-GM₂ antibody-producing clone. The abscissa indicates the relativeamount of FUT8 mRNA to the amount of β-actin mRNA when the amount in theparent clone KM968 was assumed to be 100, and the ordinate indicateseach clones. In the drawing, black and outline bars indicate therelative amount of FUT8 mRNA of the parent clone and of each clonesobtained by introducing the GMD-targeting siRNA and FUT8-targeting siRNAco-expression vector, respectively, when the amount in the parent cloneKM968 was assumed to be 100.

FIG. 23 shows the relative amount of GMD mRNA expressed in each cloneobtained by introducing the mouse GMD-targeting siRNA and FUT8-targetingsiRNA co-expression vector into clone NS0/2160 which was NS0cell-derived anti-CCR4 antibody-producing clone. The abscissa indicatesthe relative amount of GMD mRNA to the amount of β-actin mRNA when thatamount in the parent clone NS0/2160 was assumed to be 100, and theordinate indicates each clones. In the drawing, black and outline barsindicate the relative amount of GMD mRNA of the parent clone and of eachclones obtained by introducing the GMD-targeting siRNA andFUT8-targeting siRNA co-expression vector, respectively, when the amountin the parent clone NS0/2160 was assumed to be 100.

FIG. 24 shows the relative amount of FUT8 mRNA expressed in each cloneobtained by introducing mouse GMD-targeting siRNA and FUT8-targetingsiRNA co-expression vector into clone NS0/2160 which was NS0cell-derived anti-CCR4 antibody-producing clone. The abscissa indicatesthe relative amount of GMD mRNA to the amount of β-actin mRNA when theamount in the parent clone NS0/2160 was assumed to be 100 and theordinate indicates each clones. In the drawing, black and outline barsindicate the relative amount of FUT8 mRNA of the parent clone and ofeach clones obtained by introducing the GMD-targeting siRNA andFUT8-targeting siRNA co-expression vector, respectively, when the amountin the parent clone NS0/2160 was assumed to be 100.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a cell into which an RNA capable ofsuppressing the function of an enzyme catalyzing a reaction whichconverts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose is introduced;a process for producing a glycoprotein using the cell; an RNA used forpreparing the cell; a DNA corresponding to the RNA; and a vectorcomprising the DNA and its complementary DNA.

Also, the present invention provides a cell into which an RNA capable ofsuppressing the function of an enzyme relating to modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complextype N-glycoside-linked sugar chain, and an RNA capable of suppressingthe function of an enzyme protein relating to synthesis of anintracellular sugar nucleotide, GDP-fucose, or an RNA capable ofsuppressing the function of a protein relating to transport of anintracellular sugar nucleotide, GDP-fucose, to the Golgi body areintroduced; and a process for producing a glycoprotein composition usingthe cell.

Furthermore, the present invention provides a DNA comprising a DNAcorresponding to an RNA capable of suppressing the function of an enzymerelating to modification of a sugar chain in which 1-position of fucoseis bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain andits complementary DNA, and a DNA corresponding to an RNA capable ofsuppressing the function of an enzyme relating to synthesis of anintracellular sugar nucleotide, GDP-fucose, and or an RNA capable ofsuppressing the function of a protein relating to transport of anintracellular sugar nucleotide, GDP-fucose, to the Golgi body and itscomplementary DNA; a vector comprising the DNA; a cell into which thevector is introduced; a cell into which a vector comprising a DNAcorresponding to an RNA capable of suppressing the function of an enzymerelating to modification of a sugar chain in which 1-position of fucoseis bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain andits complementary DNA, and a vector comprising a DNA corresponding to anRNA capable of suppressing the function of an enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, and or anRNA capable of suppressing the function of a protein relating totransport of an intracellular sugar nucleotide, GDP-fucose, to the Golgibody and its complementary DNA are introduced; and a process forproducing a glycoprotein composition using the cell.

In the present invention, the enzyme relating to synthesis of anintracellular sugar nucleotide, GDP-fucose (hereinafter also referred toas “GDP-fucose synthase”) may be any enzyme, so long as it is an enzymerelating to synthesis of an intracellular sugar nucleotide, GDP-fucose,as a supply source of fucose to a sugar chain in a cell. Also, theGDP-fucose synthase in the present invention includes an enzyme whichhas influence on synthesis of an intracellular sugar nucleotide,GDP-fucose, and the like.

The intracellular GDP-fucose is supplied by a de novo synthesis pathwayor a salvage synthesis pathway. Thus, all enzymes and proteins relatingto the synthesis pathways are included in the GDP-fucose synthase.

The GDP-fucose synthase relating to the de novo synthesis pathwayincludes an enzyme catalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose, an enzyme catalyzing a reaction whichconverts GDP-4-keto,6-deoxy-GDP-mannose into GDP-fucose, and the like.

As the enzyme catalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose in the present invention, an enzymecatalyzing a dehydration reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose is preferably used. The enzyme catalyzinga dehydration reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose includes GDP-mannose 4,6-dehydratase.

The enzyme catalyzing a reaction which convertsGDP-4-keto,6-deoxy-GDP-mannose into GDP-fucose in the present inventionincludes an enzyme catalyzing a reaction which convertsGDP-4-keto,6-deoxy-GDP-mannose into GDP-4-keto,6-deoxy-GDP-fucose, anenzyme catalyzing a reaction which reduces the 4-position ofGDP-4-keto,6-deoxy-GDP-fucose and the like. Specific examples includeGDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase having enzymeactivity of catalyzing a relation which convertsGDP-4-keto,6-deoxy-GDP-mannose into GDP-4-keto,6-deoxy-GDP-fucose andcatalyzing a reaction which reduces the 4-position ofGDP-4-keto,6-deoxy-GDP-fucose, and the like.

The GDP-fucose synthase relating to the salvage synthesis pathwayincludes GDP-beta-L-fucose pyrophosphorylase, fucokinase and the like.

As the enzyme which has influence on the synthesis of an intracellularsugar nucleotide, GDP-fucose, an enzyme which has influence on theactivity of the above GDP-fucose synthase and an enzyme which hasinfluence on the structure of substances as the substrate of the enzymeare also included.

The protein relating to transport of an intracellular sugar nucleotide,GDP-fucose, to the Golgi body in the present invention may be anyprotein, so long as it relates to transport of an intracellular sugarnucleotide, GDP-fucose, to the Golgi body. Specific examples includeGDP-fucose transporter and the like.

The cell into which an RNA capable of suppressing the function of anenzyme catalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose is introduced in the present inventionmay be any cell, so long as it is a cell into which an RNA capable ofsuppressing the activity or expression of an enzyme catalyzing areaction which converts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose(hereinafter also referred to as “GDP-mannose converting enzyme”).

In the present invention, GDP-mannose 4,6-dehydratase is preferably usedas the GDP-mannose converting enzyme.

In the present invention, the GDP-mannose 4,6-dehydratase includes aprotein encoded by a DNA of the following (a) to (f), a protein of thefollowing (g) to (O) and the like:

(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:8;

(b) a DNA comprising the nucleotide sequence represented by SEQ ID NO:9;

(c) a DNA comprising the nucleotide sequence represented by SEQ IDNO:10;

(d) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:8 under stringent conditions andencodes a protein having GDP-mannose 4,6-dehydratase activity;

(e) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:9 under stringent conditions andencodes a protein having GDP-mannose 4,6-dehydratase activity;

(f) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:10 under stringent conditions andencodes a protein having GDP-mannose 4,6-dehydratase activity;

(g) a protein comprising the amino acid sequence represented by SEQ IDNO:11;

(h) a protein comprising the amino acid sequence represented by SEQ IDNO:12;

(i) a protein comprising the amino acid sequence represented by SEQ IDNO:13;

(j) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:11 and having GDP-mannose4,6-dehydratase activity;

(k) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:12 and having GDP-mannose4,6-dehydratase activity;

(l) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:13 and having GDP-mannose4,6-dehydratase activity;

(m) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:11 andhaving GDP-mannose 4,6-dehydratase activity;

(n) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:12 andhaving GDP-mannose 4,6-dehydratase activity;

(o) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:13 andhaving GDP-mannose 4,6-dehydratase activity.

Also, the DNA encoding the amino acid sequence of GDP-mannose4,6-dehydratase includes a DNA comprising the nucleotide sequencerepresented by any one of SEQ ID NOs:8 to 10, a DNA which hybridizeswith a DNA consisting of the nucleotide sequence represented by any oneof SEQ ID NOs:8 to 10 under stringent conditions and encodes an aminoacid sequence having GDP-mannose 4,6-dehydratase activity, and the like.

Also, the present invention relates to a cell into which an RNA capableof suppressing the function of an enzyme relating to modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complextype N-glycoside-linked sugar chain, and an RNA capable of suppressingthe function of an enzyme relating to synthesis of an intracellularsugar nucleotide, GDP-fucose, or an RNA capable of suppressing thefunction of a protein relating to transport of an intracellular sugarnucleotide, GDP-fucose, to the Golgi body are introduced.

The cell into which an RNA capable of suppressing the function of anenzyme relating to modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain, andan RNA capable of suppressing the function of an enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, or an RNAcapable of suppressing the function of a protein relating to transportof an intracellular sugar nucleotide, GDP-fucose, to the Golgi body areintroduced in the present invention may be any cell, so long as it is acell into which an RNA capable of suppressing the activity or expressionof an enzyme relating to modification of a sugar chain in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in the complex type N-glycoside-linkedsugar chain (hereinafter also referred to as “α1,6-fucose modifyingenzyme”), and an RNA capable of suppressing the activity or expressionof an enzyme relating to synthesis of an intracellular sugar nucleotide,GDP-fucose, or an RNA capable of suppressing the activity or expressionof a protein relating to transport of an intracellular sugar nucleotide,GDP-fucose, to the Golgi body are introduced. A cell into which an RNAcapable of suppressing the activity or expression of an α1,6-fucosemodifying enzyme and an RNA capable of suppressing the activity orexpression of an enzyme relating to synthesis of an intracellular sugarnucleotide, GDP-fucose, are introduced is preferred, and a cell intowhich an RNA capable of suppressing the activity or expression of anα1,6-fucose modifying enzyme and an RNA capable of suppressing theactivity or expression of an enzyme catalyzing a reaction which convertsGDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose (GDP-mannose convertingenzyme) is more preferred.

In the present invention, the α1,6-fucose modifying enzymes include anyenzyme, so long as it is an enzyme relating to modification of a sugarchain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complextype N-glycoside-linked sugar chain. Specific examples of theα1,6-fucose modifying enzyme include α1,6-fucosyltransferase,α-L-fucosidase and the like.

The α1,6-fucosyltransferase includes a protein encoded by a DNA of thefollowing (a) to (h), a protein of the following (i) to (t), and thelike:

(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO:1;

(b) a DNA comprising the nucleotide sequence represented by SEQ ID NO:2;

(c) a DNA comprising the nucleotide sequence represented by SEQ ID NO:3;

(d) a DNA comprising the nucleotide sequence represented by SEQ ID NO:4;

(e) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:1 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity;

(f) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:2 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity;

(g) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:3 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity;

(h) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:4 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity;

(i) a protein comprising the amino acid sequence represented by SEQ IDNO:5;

(j) a protein comprising the amino acid sequence represented by SEQ IDNO:6;

(k) a protein comprising the amino acid sequence represented by SEQ IDNO:7;

(l) a protein comprising the amino acid sequence represented by SEQ IDNO:84;

(m) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:5 and havingα1,6-fucosyltransferase activity;

(n) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:6 and havingα1,6-fucosyltransferase activity;

(o) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:7 and havingα1,6-fucosyltransferase activity;

(p) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO: 84 and havingα1,6-fucosyltransferase activity;

(q) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:5 andhaving α1,6-fucosyltransferase activity;

(r) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:6 andhaving α1,6-fucosyltransferase activity;

(s) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:7 andhaving α1,6-fucosyltransferase activity;

(t) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:84 andhaving α1,6-fucosyltransferase activity.

Also, the DNA encoding the amino acid sequence of theα1,6-fucosyltransferase includes a DNA comprising the nucleotidesequence represented by any one of SEQ ID NOs:1 to 4, a DNA whichhybridizes with a DNA consisting of the nucleotide sequence representedby any one of SEQ ID NOs:1 to 4 under stringent conditions and encodesan amino acid sequence having α1,6-fucosyltransferase activity, and thelike.

In the present invention, a DNA which can hybridize under stringentconditions is a DNA obtained, e.g., by a method such as colonyhybridization, plaque hybridization or Southern blot hybridization usinga DNA such as the DNA having the nucleotide sequence represented by anyone of SEQ ID NOs:1 to 4 and 8 to 10 or a partial fragment thereof asthe probe, and specifically includes a DNA which can be identified bycarrying out hybridization at 65° C. in the presence of 0.7 to 1.0 mol/Lsodium chloride using a filter to which colony- or plaque-derived DNAare immobilized, and then washing the filter at 65° C. using 0.1 to2×SSC solution (composition of the 1×SSC solution comprising 150 mmol/Lsodium chloride and 15 mmol/L sodium citrate). The hybridization can becarried out in accordance with the methods described, e.g., in MolecularCloning, A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press (1989) (hereinafter referred to as “Molecular Cloning,Second Edition”), Current Protocols in Molecular Biology, John Wiley &Sons, 1987-1997 (hereinafter referred to as “Current Protocols inMolecular Biology”); DNA Cloning 1: Core Techniques, A PracticalApproach, Second Edition, Oxford University (1995); and the like. Thehybridizable DNA includes a DNA having at least 60% or more, preferably70% or more, more preferably 80% or more, still more preferably 90% ormore, far more preferably 95% or more, and most preferably 98% or more,homology with the nucleotide sequence represented by any one of SEQ IDNOs:1 to 4 and 8 to 10.

In the present invention, the protein which comprises an amino acidsequence in which one or more amino acid(s) is/are deleted, substituted,inserted and/or added in the amino acid sequence represented by any oneof SEQ ID NOs:5 to 7 and 84 and has α1,6-fucosyltransferase activity orthe protein which comprises an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by any one of SEQ ID NOs:11 to 13 andhas GDP-mannose 4,6-dehydratase activity can be obtained, e.g., byintroducing a site-directed mutation into a DNA encoding a proteinhaving the amino acid sequence represented by any one of SEQ ID NOs:5 to7 and 84 or any one of SEQ ID NOs:11 to 13, using the site-directedmutagenesis described, e.g., in Molecular Cloning, Second Edition;Current Protocols in Molecular Biology; Nucleic Acids Research, 10, 6487(1982); Proc. Natl. Acad. Sci. USA, 79, 6409 (1982); Gene, 34, 315(1985); Nucleic Acids Research, 13, 4431 (1985); Proc. Natl. Acad. Sci.USA, 82, 488 (1985); and the like. The number of amino acids to bedeleted, substituted, inserted and/or added is one or more, and thenumber is not particularly limited, but is a number which can bedeleted, substituted or added by a known technique such as thesite-directed mutagenesis, e.g., it is 1 to several tens, preferably 1to 20, more preferably 1 to 10, and most preferably 1 to 5.

In the present invention, the protein which comprises an amino acidsequence having 80% or more homology with the amino acid sequencerepresented by any one of SEQ ID NOs:11 to 13 and has GDP-mannose4,6-dehydratase activity is a protein having at least 80% or more,preferably 85% or more, more preferably 90% or more, still morepreferably 95% or more, far more preferably 97% or more, and mostpreferably 99% or more, homology with the amino acid sequencerepresented by any one of SEQ ID NOs:11 to 13 and having GDP-mannose4,6-dehydratase activity.

Also, in the present invention, the protein which comprises an aminoacid sequence having 80% or more homology with the amino acid sequencerepresented by any one of SEQ ID NOs:5 to 7 and 84 and hasα1,6-fucosyltransferase activity is a protein having at least 80% ormore, preferably 85% or more, more preferably 90% or more, still morepreferably 95% or more, far more preferably 97% or more, and mostpreferably 99% or more, homology with the amino acid sequencerepresented by any one of SEQ ID NOs:5 to 7 and 84 and havingα1,6-fucosyltransferase activity.

The number of the homology described in the present invention may be anumber calculated by using a known homology search program, unlessotherwise indicated. Regarding the nucleotide sequence, the number maybe calculated by using a default parameter in BLAST [J. Mol. Biol., 215,403 (1990)] or the like, and regarding the amino acid sequence, thenumber may be calculated by using a default parameter in BLAST2 [NucleicAcids Res., 25, 3389 (1997); Genome Res., 7, 649 (1997);http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html] orthe like.

As the default parameter, G (cost to open gap) is 5 for the nucleotidesequence and 11 for the amino acid sequence; -E (cost to extend gap) is2 for the nucleotide sequence and 1 for the amino acid sequence; -q(penalty for nucleotide mismatch) is −3; -r (reward for nucleotidematch) is 1; -e (expect value) is 10; —W (wordsize) is 11 residues forthe nucleotide sequence and 3 residues for the amino acid sequence; -y(dropoff (X) for blast extensions in bits) is 20 for blastn and 7 for aprogram other than blastn; -X (X dropoff value for gapped alignment inbits) is 15; and -Z (final X dropoff value for gapped alignment in bits)is 50 for blastn and 25 for a program other than blastn(http://www.ncbi.nlm.nih.govlblast/html/blastcgihelp.html).Additionally, the analysis software of the amino acid sequence alsoincludes FASTA [Methods in Enzymology, 183, 63(1990)], and the like.

The cell used in the present invention may be any cell, so long as itcan express a glycoprotein such as an antibody molecule. Examplesinclude an yeast, an animal cell, an insect cell, a plant cell and thelike, and an animal cell is preferred. Preferred examples of the animalcell include a CHO cell derived from a Chinese hamster ovary tissue, arat myeloma cell line YB2/3HL.P2.G11.16Ag.20 cell, a mouse myeloma cellline NS0 cell, a mouse myeloma SP2/0-Ag14 cell, a BHK cell derived froma syrian hamster kidney tissue, an antibody-producing-hybridoma cell, ahuman leukemia cell line Namalwa cell, an embryonic stem cell, afertilized egg cell, and the like.

The cell into which an RNA capable of suppressing the function of aGDP-mannose converting enzyme is introduced or the cell into which anRNA capable of suppressing the function of an enzyme relating tomodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex type N-glycoside-linked sugar chain, and an RNA capable ofsuppressing the function of an enzyme relating to synthesis of anintracellular sugar nucleotide, GDP-fucose, or an RNA capable ofsuppressing the function of a protein relating to transport of anintracellular sugar nucleotide, GDP-fucose, to the Golgi body areintroduced in the present invention (hereinafter both being referred toas “the cell of the present invention” as a whole) is resistant to alectin which recognizes a sugar chain structure in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in a N-glycoside-linked sugar chain. The parent cell intowhich the RNA is not introduced is not resistant to the lectin.

Accordingly, in the present invention, the cell of the present inventionincludes a cell such as a yeast, an animal cell, an insect cell or aplant cell which can produce a glycoprotein composition and is resistantto a lectin which recognizes a sugar chain structure in which 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingend through α-bond in a complex type N-glycoside-linked sugar chain.Examples include a hybridoma cell, a host cell for producing a humanantibody or a humanized antibody, an embryonic stem cell and fertilizedegg cell for producing a transgenic non-human animal which produces ahuman antibody, a plant callus cell for producing a transgenic plantwhich produces a human antibody, a myeloma cell, a cell derived from atransgenic non-human animal and the like which are resistant to lectinwhich recognizes a sugar chain structure in which 1-position of fucoseis bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in a complex type N-glycoside-linked sugar chain. Themyeloma cell can be used as a fusion cell for producing a hybridomacell. Also, a hybridoma cell can be produced by immunizing a transgenicnon-human animal with an antigen and using antibody-producing cells suchas spleen cells of the animal.

The cell resistant to a lectin is a cell of which growth is notinhibited when the cell is cultured by applying the lectin to a culturemedium at an effective concentration.

In the present invention, the effective concentration of the lectinwhich does not inhibit the growth can be adjusted depending on the cellline used as the parent cell, and is generally 10 μg/ml to 10.0 mg/ml,preferably 0.5 to 2.0 mg/ml. When an RNA capable of suppressing thefunction of a GDP-mannose converting enzyme is introduced into a parentcell, or when an RNA capable of suppressing the function of an enzymerelating to modification of a sugar chain in which 1-position of fucoseis bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain, andan RNA capable of suppressing the function of an enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, or an RNAcapable of suppressing the function of a protein relating to transportof an intracellular sugar nucleotide, GDP-fucose, to the Golgi body areintroduced into a parent cell, the effective concentration of the lectinis a concentration in which the parent cell cannot normally grow orhigher than the concentration, and is a concentration which ispreferably the same degree, more preferably 2 to 5 times, still morepreferably at least 10 times, and most preferably at least 20 times,higher than the concentration in which the parent cell cannot normallygrow.

The parent cell means a cell before introduction of an RNA capable ofsuppressing the function of a GDP-mannose converting enzyme or a cellbefore introduction of an RNA capable of suppressing the function of anenzyme relating to modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain, andan RNA capable of suppressing the function of an enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, or an RNAcapable of suppressing the function of a protein relating to transportof an intracellular sugar nucleotide, GDP-fucose, to the Golgi body.

Although the parent cell is not particularly limited, the followingcells are exemplified.

The parent cell of NS0 cell includes NS0 cells described in literaturessuch as BIO/TECHNOLOGY, 10, 169 (1992) and Biotechnol. Bioeng., 73, 261(2001). Also, it includes cell line NS0 (RCB 0213) registered at RIKENCell Bank, The Institute of Physical and Chemical Research, sub-celllines obtained by adapting these cell lines to various media in whichthey can grow, and the like.

The parent cell of SP2/0-Ag14 cell includes SP2/0-Ag14 cells describedin literatures such as J. Immunol., 126, 317 (1981), Nature, 276, 269(1978) and Human Antibodies and Hybridomas, 3, 129 (1992). Also, itincludes SP2/0-Ag14 cell (ATCC CRL-1581) registered at ATCC, sub-celllines obtained by adapting these cell lines to various media in whichthey can grow (ATCC CRL-1581.1), and the like.

The parent cell of CHO cell derived from Chinese hamster ovary tissueincludes CHO cells described in literatures such as Journal ofExperimental Medicine, 108, 945 (1958), Proc. Natl. Acad. Sci. USA, 60,1275 (1968), Genetics, 55, 513 (1968), Chromosoma, 41, 129 (1973),Methods in Cell Science, 18, 115 (1996), Radiation Research, 148, 260(1997), Proc. Natl. Acad. Sci. USA, 77, 4216 (1980), Proc. Natl. Acad.Sci. USA, 60, 1275 (1968), Cell, 6, 121 (1975) and Molecular CellGenetics, Appendix I, II (p. 883-900). Also, it includes cell lineCHO-K1 (ATCC CCL-61), cell line DUXB11 (ATCC CRL-9096) and cell linePro-5 (ATCC CRL-1781) registered at ATCC, commercially available cellline CHO-S (Cat # 11619 of Lifetechnologies), sub-cell lines obtained byadapting these cell lines to various media in which they can grow, andthe like.

The parent cell of a rat myeloma cell line YB2/3HL.P2.G11.16Ag.20 cellincludes cell lines established from Y3/Ag1.2.3 cell (ATCC CRL-1631)such as YB2/3HL.P2.G11.16Ag.20 cell described in literatures such as J.Cell. Biol., 93, 576 (1982) and Methods Enzymol., 73B, 1 (1981). Also,it includes YB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL-1662) registered atATCC, sub-lines obtained by adapting these cell lines to various mediain which they can grow, and the like.

In addition to the above, the parent cell used in the present inventionincludes a human leukemia cell line NM-F9 cell (DSM ACC2605,WO05/17130), a human embryonic retinal cell line PER.C6 cell (ECACC No.96022940, U.S. Pat. No. 6,855,544), sub-lines obtained by adapting thesecell lines to various media in which they can grow, and the like.

The lectin which recognizes a sugar chain structure in which 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingend through α-bond in the complex type N-glycoside-linked sugar chainmay be any lectin, so long as it can recognize the sugar chainstructure. Examples include a Lens culinaris agglutinin LCA (lentilagglutinin derived from Lens culinaris), a Pisum sativum agglutinin PSA(pea lectin derived from Pisum sativum), a Vicia faba agglutinin VFA(agglutinin derived from Vicia faba), an Aleuria aurantia lectin AAL(lectin derived from Aleuria aurantia) and the like.

In the present invention, the RNA capable of suppressing the function ofan enzyme relating to modification of a sugar chain in which 1-positionof fucose is bound to 6-position of N-acetylglucosamine in the reducingend through α-bond in the complex type N-glycoside-linked sugar chain,and the RNA capable of suppressing the function of an enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, or the RNAcapable of suppressing the function of a protein relating to transportof an intracellular sugar nucleotide, GDP-fucose, to the Golgi body maybe any RNAs, so long as they comprise an RNA and its complementary RNAand is a double-stranded RNA capable of decreasing the amount of mRNA ofan α1,6-fucose modifying enzyme and a double-stranded RNA capable ofdecreasing the amount of mRNA of an enzyme relating to synthesis of anintracellular sugar nucleotide, GDP-fucose, or the amount of mRNA of aprotein relating to transport of an intracellular sugar nucleotide,GDP-fucose, to the Golgi body, respectively. Regarding the length of theRNA, a continuous RNA of 10 to 40, preferably 10 to 30, and morepreferably 15 to 30, is exemplified.

The RNA capable of suppressing the function of an enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, or the RNAcapable of suppressing the function of a protein relating to transportof an intracellular sugar nucleotide, GDP-fucose, to the Golgi body ispreferably an RNA capable of decreasing the amount of mRNA of aGDP-mannose converting enzyme.

Examples include:

(a) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:8;

(b) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:9; and

(c) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:10.

Preferred examples include:

(1) an RNA comprising the nucleotide sequence represented by SEQ IDNO:37;

(2) an RNA consisting of a nucleotide sequence in which one or a severalnucleotide(s) is/are deleted, substituted, inserted and/or added in thenucleotide sequence represented by SEQ ID NO:37 and having activity ofsuppressing the function of a GDP-mannose converting enzyme;

(3) an RNA comprising the nucleotide sequence represented by SEQ IDNO:57;

(4) an RNA consisting of a nucleotide sequence in which one or a severalnucleotide(s) is/are deleted, substituted, inserted and/or added in thenucleotide sequence represented by SEQ ID NO:57 and having activity ofsuppressing the function of a GDP-mannose converting enzyme;

(5) an RNA comprising the nucleotide sequence represented by SEQ IDNO:58;

(6) an RNA consisting of a nucleotide sequence in which one or a severalnucleotide(s) is/are deleted, substituted, inserted and/or added in thenucleotide sequence represented by SEQ ID NO:58 and having activity ofsuppressing the function of a GDP-mannose converting enzyme.

In the RNAs of the above-described (a) to (c) and (1) to (6), it ispreferred that the RNAs in (a), (1) and (2) are used in a parent cellderived from a hamster, the RNAs in (b), (3) and (4) are used in aparent cell derived from a human, and the RNAs in (c), (5) and (6) areused in a parent cell line derived from a mouse, as an RNA capable ofsuppressing the function of a GDP-mannose converting enzyme.

In the present invention, the RNA capable of suppressing the function ofan α1,6-fucose modifying enzyme is preferably an RNA capable ofdecreasing the amount of mRNA of an α1,6-fucose modifying enzyme. Thelength of the RNA is, for example, 10 to 40, preferably 10 to 30 andmore preferably 15 to 30.

Examples include:

(d) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:1;

(e) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:2;

(f) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:3;

(g) an RNA corresponding to a DNA consisting of a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:4.

Preferred examples include:

(7) an RNA comprising the nucleotide sequence represented by SEQ IDNO:14;

(8) an RNA consisting of a nucleotide sequence in which one or a severalnucleotide(s) is/are deleted, substituted, inserted and/or added in thenucleotide sequence represented by SEQ ID NO:14 and having activity ofsuppressing the function of an α1,6-fucose modifying enzyme;

(9) an RNA comprising the nucleotide sequence represented by SEQ IDNO:15;

(10) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:15 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(11) an RNA comprising the nucleotide sequence represented by SEQ IDNO:16;

(12) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:16 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(13) an RNA comprising the nucleotide sequence represented by SEQ IDNO:17;

(14) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:17 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(15) an RNA comprising the nucleotide sequence represented by SEQ IDNO:18;

(16) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:18 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(17) an RNA comprising the nucleotide sequence represented by SEQ IDNO:19;

(18) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:19 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(19) an RNA comprising the nucleotide sequence represented by SEQ IDNO:20;

(20) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:20 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(21) an RNA comprising the nucleotide sequence represented by SEQ IDNO:21;

(22) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:21 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(23) an RNA comprising the nucleotide sequence represented by SEQ IDNO:22;

(24) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:22 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(25) an RNA comprising the nucleotide sequence represented by SEQ IDNO:23;

(26) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:23 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(27) an RNA comprising the nucleotide sequence represented by SEQ IDNO:24;

(28) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:24 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(29) an RNA comprising the nucleotide sequence represented by SEQ IDNO:25;

(30) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:25 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(31) an RNA comprising the nucleotide sequence represented by SEQ IDNO:26;

(32) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:26 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(33) an RNA comprising the nucleotide sequence represented by SEQ IDNO:27;

(34) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:27 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(35) an RNA comprising the nucleotide sequence represented by SEQ IDNO:28;

(36) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:28 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(37) an RNA comprising the nucleotide sequence represented by SEQ IDNO:29;

(38) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:29 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(39) an RNA comprising the nucleotide sequence represented by SEQ IDNO:30;

(40) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:30 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(41) an RNA comprising the nucleotide sequence represented by SEQ IDNO:31;

(42) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:31 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(43) an RNA comprising the nucleotide sequence represented by SEQ IDNO:32;

(44) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:32 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(45) an RNA comprising the nucleotide sequence represented by SEQ IDNO:33;

(46) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:33 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(47) an RNA comprising the nucleotide sequence represented by SEQ IDNO:34;

(48) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotides are more nucleotide(s) is/are deleted, substituted,inserted and/or added in the nucleotide sequence represented by SEQ IDNO:34 and having activity of suppressing the function of an α1,6-fucosemodifying enzyme;

(49) an RNA comprising the nucleotide sequence represented by SEQ IDNO:35;

(50) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:35 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(51) an RNA comprising the nucleotide sequence represented by SEQ IDNO:85;

(52) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:85 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(53) an RNA comprising the nucleotide sequence represented by SEQ IDNO:86;

(54) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:86 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(55) an RNA comprising the nucleotide sequence represented by SEQ IDNO:87;

(56) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:87 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(57) an RNA comprising the nucleotide sequence represented by SEQ IDNO:88;

(58) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:88 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme;

(59) an RNA comprising the nucleotide sequence represented by SEQ IDNO:89;

(60) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by SEQ ID NO:89 and havingactivity of suppressing the function of an α1,6-fucose modifying enzyme/

In the RNAs of the above-described (d) to (g) and (7) to (60), it ispreferred that the RNAs of (d) and (7) to (26) are used as a parent cellderived from a hamster, the RNAs of (e), (11), (12), (23) to (28), (33)to (39), (41), (42), (45), (46), (51), (52), (57) and (58) are used as aparent cell derived from a mouse, the RNA of (f), (7), (8), (11), (12),(19), (20), (23) to (26), (33), (34), (39) to (42), (49), (50), (55) and(56) are used as a parent cell derived from a rat, and the RNAs of (g),(23) to (26), (29) to (34), (37), (38), (43), (44), (47), (48), (53),(54), (59) and (60) are used as a parent cell derived from a human, asan RNA capable of suppressing the function of an α1,6-fucose modifyingenzyme.

The nucleotide sequence in which one or a several nucleotide(s) is/aredeleted, substituted, inserted and/or added is, for example, anucleotide sequence in which one or a several nucleotide(s) is/aredeleted, substituted, inserted and/or added in the nucleotide sequencerepresented by any one of SEQ ID NOs:14 to 37, 57, 58 and 85 to 89. Adouble-stranded RNA formed by the deletion, substitution, insertionand/or addition of the nucleotide may be an RNA in which the nucleotideis deleted, substituted, inserted and/or added in only one of thestrands. That is, the double-stranded RNA may not always be a completecomplementary strand, so long as the effect of the present invention isobtained.

Also, the present invention relates to a vector comprising a DNAcorresponding to an RNA capable of suppressing the function of an enzymerelating to modification of a sugar chain in which 1-position of fucoseis bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain andits complementary DNA, and a DNA corresponding to an RNA capable ofsuppressing the function of an enzyme relating to synthesis of anintracellular sugar nucleotide, GDP-fucose, and its complementary DNA ora DNA corresponding to an RNA capable of suppressing the function of aprotein relating to transport of an intracellular sugar nucleotide,GDP-fucose, to the Golgi body and its complementary DNA; and a vectorcomprising the DNA.

The DNA comprising a DNA corresponding to an RNA capable of suppressingthe function of an enzyme relating to modification of a sugar chain inwhich 1-position of fucose is bound to 6-position of N-acetylglucosaminein the reducing end through α-bond in the complex typeN-glycoside-linked sugar chain and its complementary DNA, and a DNAcorresponding to an RNA capable of suppressing the function of an enzymerelating to synthesis of an intracellular sugar nucleotide, GDP-fucose,and its complementary DNA or a DNA corresponding to an RNA capable ofsuppressing the function of a protein relating to transport of anintracellular sugar nucleotide, GDP-fucose, to the Golgi body and itscomplementary DNA in the present invention may be any DNA, so long as itis a DNA comprising a DNA corresponding to an RNA capable of suppressingthe activity or expression of an α1,6-fucose modifying enzyme and itscomplementary DNA, and a DNA corresponding to an RNA capable ofsuppressing the activity or expression of an enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, and itscomplementary DNA or the DNA corresponding to an RNA capable ofsuppressing the activity or expression of a protein relating totransport of an intracellular sugar nucleotide, GDP-fucose, to the Golgibody and its complementary DNA. A DNA comprising a DNA corresponding toan RNA capable of suppressing the activity or expression of anα1,6-fucose modifying enzyme and its complementary DNA, and a DNAcorresponding to an RNA capable of suppressing the activity orexpression of an enzyme relating to synthesis of an intracellular sugarnucleotide, GDP-fucose, and its complementary DNA is preferred, and aDNA comprising a DNA corresponding to an RNA capable of suppressing theactivity or expression of an α1,6-fucose modifying enzyme and itscomplementary DNA, and a DNA corresponding to an RNA capable ofsuppressing the function of a GDP-mannose converting enzyme and itscomplementary DNA is more preferred.

The vector of the present invention may be any vector, so long as it isa vector comprising the above-described DNA comprising a DNAcorresponding to an RNA capable of suppressing the function of an enzymerelating to modification of a sugar chain in which 1-position of fucoseis bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex N-glycoside-linked sugar chain and itscomplementary DNA, and a DNA corresponding to an RNA capable ofsuppressing the function of an enzyme relating to synthesis of anintracellular sugar nucleotide, GDP-fucose, and its complementary DNA ora DNA corresponding to an RNA capable of suppressing the function of aprotein relating to transport of an intracellular sugar nucleotide,GDP-fucose, to the Golgi body and its complementary DNA in the presentinvention.

The vector of the present invention may be constructed, for example, byinserting, into a commercially available siRNA expression vector, theabove-described DNA comprising a DNA corresponding to an RNA capable ofsuppressing the function of an enzyme relating to modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complextype N-glycoside-linked sugar chain and its complementary DNA, and a DNAcorresponding to an RNA capable of suppressing the function of an enzymerelating to synthesis of an intracellular sugar nucleotide, GDP-fucose,and its complementary DNA or a DNA corresponding to an RNA capable ofsuppressing the function of a protein relating to transport of anintracellular sugar nucleotide, GDP-fucose, to the Golgi body and itscomplementary DNA.

The vector of the present invention may be constructed, as one vector,by inserting, into a vector, a DNA corresponding to an RNA capable ofsuppressing the function of an enzyme relating to modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complextype N-glycoside-linked sugar chain and its complementary DNA so as tobe transcribed by a respectively independent promoter, and by inserting,into a vector, a DNA corresponding to an RNA capable of suppressing thefunction of an enzyme relating to synthesis of an intracellular sugarnucleotide, GDP-fucose, and its complementary DNA or a DNA correspondingto an RNA capable of suppressing the function of a protein relating totransport of an intracellular sugar nucleotide, GDP-fucose, to the Golgibody and its complementary DNA so as to be transcribed by a respectivelyindependent promoter. However, in order to easily form a double-strandedRNA, it is preferred that a DNA in which a DNA corresponding to an RNAcapable of suppressing the function of an α1,6-fucose modifying enzymeand its complementary DNA are linked via a linker sequence is prepared,the DNA is inserted into a vector so as to be transcribed by anindependent promoter, a DNA in which a DNA corresponding to an RNAcapable of suppressing the function of an enzyme relating to synthesisof an intracellular sugar nucleotide, GDP-fucose, and its complementaryDNA are linked via a linker sequence or a DNA in which a DNAcorresponding to an RNA capable of suppressing the function of a proteinrelating to transport of an intracellular sugar nucleotide, GDP-fucose,to the Golgi body and its complementary DNA are linked via a linkersequence is prepared, and the DNA is inserted into the vector so as tobe transcribed by an independent promoter to thereby construct thevector of the present invention as one vector.

Furthermore, a DNA in which a DNA corresponding to an RNA capable ofsuppressing the function of an α1,6-fucose modifying enzyme and itscomplementary DNA are linked via a linker sequence, and a DNA in which aDNA corresponding to an RNA capable of suppressing the function of anenzyme relating to synthesis of an intracellular sugar nucleotide,GDP-fucose, and its complementary DNA are linked via a linker or a DNAin which a DNA corresponding to an RNA capable of suppressing thefunction of a protein relating to transport of an intracellular sugarnucleotide, GDP-fucose, to the Golgi body and its complementary DNA arelinked via a linker may be inserted into a vector under one promoter soas to be transcribed to one continuous RNA.

The two independent promoters are either promoters of the same kind orpromoters of different kinds. The directions of the two independentpromoters are either the same direction or opposite directions. As thetwo independent promoters in the vector of the present invention, it ispreferred that promoters of different kinds are positioned toward thesame direction.

The promoter may be any promoter, so long as it is a promoter capable offunctioning in a parent cell. When an animal cell is used as a parentcell, for example, a polymerase III promoter such as U6 promoter, H1promoter and tRNA promoter can be used.

The nucleotide sequence used as the linker may be any sequence, so longas it is a sequence used for forming a double-stranded RNA. A sequencecapable of expressing a hairpin-type siRNA in which an RNA and itscomplementary RNA are linked via a loop of about 2 to 10 nucleotides tothereby form a double-stranded RNA is preferred.

The DNA in which a DNA corresponding to an RNA capable of suppressingthe function of an α1,6-fucose modifying enzyme and its complementaryRNA and its complementary DNA are linked via a linker sequence includesa DNA selected from the nucleotide sequences represented by SEQ IDNOs:90, 91, 94 and 95.

The DNA in which a DNA and its complementary DNA corresponding to an RNAcapable of suppressing the function of an enzyme relating to synthesisof an intracellular sugar nucleotide, GDP-fucose, and its complementaryRNA, respectively, are linked via a linker sequence is preferably a DNAin which a DNA and its complementary DNA corresponding to an RNA capableof suppressing the function of a GDP-mannose converting enzyme and itscomplementary RNA, respectively, are linked via a linker sequence. TheDNA in which a DNA and its complementary DNA corresponding to an RNAcapable of suppressing the function of a GDP-mannose converting enzymeand its complementary RNA, respectively, are linked via a linkersequence is a DNA selected from the nucleotide sequences represented bySEQ ID NOs:92, 93 and 96.

In the vector of the present invention, the DNA in which a DNA and itscomplementary DNA corresponding to an RNA capable of suppressing thefunction of an α1,6-fucose modifying enzyme and its complementary RNA,respectively, are linked via a linker sequence and the DNA in which aDNA and its complementary DNA corresponding to an RNA capable ofsuppressing the function of an enzyme relating to synthesis of anintracellular sugar nucleotide, GDP-fucose, and its complementary RNA,respectively, are linked via a linker sequence or the DNA in which a DNAand its complementary DNA corresponding to an RNA capable of suppressingthe function of a protein relating to transport of an intracellularsugar nucleotide, GDP-fucose, to the Golgi body and its complementaryRNA, respectively, are linked via a linker sequence are preferablyselected as an effective combination depending on the animal species ofthe parent cell to be introduced. Specifically, when the parent cell isderived from a hamster, a combination of a DNA represented by SEQ IDNO:90 and a DNA represented by SEQ ID NO:92 or a combination of a DNArepresented by SEQ ID NO:91 and a DNA represented by SEQ ID NO:92 ispreferably used.

When the parent cell is derived from a mouse, a combination of a DNArepresented by SEQ ID NO:90 and a DNA represented by SEQ ID NO:93 or acombination of a DNA represented by SEQ ID NO:91 and a DNA representedby SEQ ID NO:93 is preferably used.

When the parent cell is derived from a human, a combination of a DNArepresented by SEQ ID NO:94 and a DNA represented by SEQ ID NO:96 or acombination of a DNA represented by SEQ ID NO:95 and a DNA representedby SEQ ID NO:96 is preferably used.

The cell into which the vector of the present invention is introduced isincluded in the cell of the present invention.

Also, the cell of the present invention includes a cell obtained byinserting, into two kinds of independent vectors, the above-describedDNA corresponding to an RNA capable of suppressing the function of anenzyme relating to modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain andits complementary DNA, and the DNA corresponding to an RNA capable ofsuppressing the function of an enzyme relating to synthesis of anintracellular sugar nucleotide, GDP-fucose, and its complementary DNA orthe DNA corresponding to an RNA capable of suppressing the function of aprotein relating to transport of an intracellular sugar nucleotide,GDP-fucose, to the Golgi body and its complementary DNA andco-introducing the two kinds of independent vectors into a cell.

The cell of the present invention is capable of producing a glycoproteinhaving a sugar chain structure having no fucose and having highphysiological activity. That is, the present invention can provide aprocess for producing a glycoprotein having higher physiologicalactivity than a glycoprotein produced by its parent cell.

Examples of the glycoprotein having high physiological activity due tothe sugar chain structure having no fucose include a glycoprotein havingimproved affinity with a receptor, a glycoprotein having improvedhalf-life in blood, a glycoprotein in which its tissue distributionafter administration into blood is changed, and a glycoprotein in whichits interaction with a protein necessary for expressing pharmacologicalactivity is improved, due to the sugar chain structure having no fucose.

Accordingly, the glycoprotein in the present invention may be anyglycoprotein, so long as it is a glycoprotein in which a producedprotein has a sugar chain structure to which fucose binds when it isproduced by the parent cell. Examples include an antibody,erythropoietin, thrombopoietin, tissue type plasminogen activator,prourokinase, thrombomodulin, antithrombin III, protein C, bloodcoagulation factor VII, blood coagulation factor VIII, blood coagulationfactor IX, blood coagulation factor X, gonadotropic hormone,thyroid-stimulating hormone, epidermal growth factor (EGF), hepatocytegrowth factor (HGF), keratinocyte growth factor, activin, bonemorphogenetic factor, stem cell factor (SCF), interferon-α,interferon-β, interferon-γ, interleukin-2, interleukin-6,interleukin-10, interleukin-11, soluble interleukin-4 receptor, tumornecrosis factor-α, DNase I, galactosidase, α-glucosidase,glucocerebrosidase and the like.

Examples of a glycoprotein having remarkably improved physiologicalactivity due to the sugar chain structure to which no fucose bindsinclude an antibody composition.

That is, the cell of the present invention can produce an antibodycomposition having higher ADCC activity than that of an antibodycomposition produced by a parent cell.

Furthermore, the cell of the present invention can produce an antibodycomposition wherein among the total complex type N-glycoside-linkedsugar chains bound to the Fc region in the antibody composition, theratio of sugar chains in which fucose is not bound toN-acetylglucosamine in the reducing end in the sugar chain is higherthan that of an antibody composition produced by a parent cell.

In the present invention, the antibody composition is a compositionwhich comprises an antibody molecule having a complex typeN-glycoside-linked sugar chain in the Fc region.

The antibody is a tetramer in which two molecules of each of twopolypeptide chains, a heavy chain and a light chain (hereinafterreferred to as “H chain” and “L chain”, respectively) are respectivelyassociated. Each of about a quarter of the N-terminal side of the Hchain and about a half of the N-terminal side of the L chain (more than100 amino acids for each) is called a variable region (hereinafterreferred to as “V region”) which is rich in diversity and directlyrelates to the binding with an antigen. The greater part of the moietyother than the V region is called a constant region (hereinafterreferred to as “C region”). Based on homology with the C region,antibody molecules are classified into classes IgG, IgM, IgA, IgD andIgE.

Also, the IgG class is further classified into subclasses IgG1 to IgG4based on homology of the C region.

The H chain is divided into four immunoglobulin domains, an antibody Hchain V region (hereinafter referred to as “VH”), an antibody H chain Cregion 1 (hereinafter referred to as “CH1”), an antibody H chain Cregion 2 (hereinafter referred to as “CH2”) and an antibody H chain Cregion 3 (hereinafter referred to as “CH3”), from its N-terminal, and ahighly flexible peptide region called hinge region is present betweenCH1 and CH2 to divide CH1 and CH2. A structural unit comprising CH2 andCH3 in the downstream of the hinge region is called Fc region to which acomplex type N-glycoside-linked sugar chain is bound. The Fc region is aregion to which an Fc receptor, a complement and the like are bound(Immunology Illustrated, the Original, 5th edition, published on Feb.10, 2000, by Nankodo; Handbook of Antibody Technology (Kotai KogakuNyumon), 1st edition on Jan. 25, 1994, by Chijin Shokan).

Sugar chains of glycoproteins such as an antibody are roughly classifiedinto two types, namely a sugar chain which binds to asparagine(N-glycoside-linked sugar chain) and a sugar chain which binds toserine, threonine or the like (O-glycoside-linked sugar chain), based onthe binding form to the protein moiety. The N-glycoside-linked sugarchains have a basic common core structure shown by the following formula(I) [Biochemical Experimentation Method 23—Method for StudyingGlycoprotein Sugar Chain (Gakujutsu Shuppan Center), edited by ReikoTakahashi (1989)]:

In formula (I), the sugar chain terminus which binds to asparagine iscalled a reducing end, and the opposite side is called a non-reducingend.

The N-glycoside-linked sugar chain may be any N-glycoside-linked sugarchain, so long as it comprises the core structure of formula (I).Examples include a high mannose type in which mannose alone binds to thenon-reducing end of the core structure; a complex type in which thenon-reducing end side of the core structure comprises one or pluralityof parallel branches of galactose-N-acetylglucosamine (hereinafterreferred to as “Gal-GlcNAc”) and the non-reducing end side of Gal-GlcNAcfurther comprises a structure of sialic acid, bisectingN-acetylglucosamine or the like; a hybrid type in which the non-reducingend side of the core structure comprises branches of both of the highmannose type and complex type; and the like.

Since the Fc region in the antibody molecule comprises regions to whichN-glycoside-linked sugar chains are separately bound, two sugar chainsare bound per one antibody molecule. Since the N-glycoside-linked sugarchain which binds to an antibody molecule includes any sugar chainhaving the core structure represented by formula (I), there are a numberof combinations of sugar chains for the two N-glycoside-linked sugarchains which bind to the antibody.

Accordingly, in the present invention, an antibody composition preparedby using a cell into which an RNA capable of suppressing the function ofa GDP-mannose converting enzyme or an antibody composition prepared byusing a cell into which an RNA capable of suppressing the function of anenzyme relating to modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain, andan RNA capable of suppressing the function of an enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, or an RNAcapable of suppressing the function of a protein relating to transportof an intracellular sugar nucleotide, GDP-fucose, to the Golgi body areintroduced may comprise an antibody molecule having the same sugar chainstructure or an antibody molecule having different sugar chainstructures, so long as the effect of the present invention can beobtained.

The ratio of sugar chains in which fucose is not bound toN-acetylglucosamine in the reducing end in the sugar chain among thetotal complex type N-glycoside-linked sugar chains bound to the Fcregion contained in the antibody composition (hereinafter referred tothe “ratio of sugar chains of the present invention”) is a ratio of thenumber of sugar chains in which fucose is not bound toN-acetylglucosamine in the reducing end in the sugar chains to the totalnumber of the complex type N-glycoside-linked sugar chains bound to theFc region contained in the composition.

The sugar chain in which fucose is not bound to N-acetylglucosamine inthe reducing end in the complex type N-glycoside-linked sugar chain is asugar chain in which fucose is not bound to N-acetylglucosamine in thereducing end through α-bond in the complex type N-glycoside-linked sugarchain. Specifically, sugar chain in which fucose is not bound toN-acetylglucosamine in the reducing end through α-bond in the complextype N-glycoside-linked sugar chain is mentioned.

The higher the ratio of sugar chains of the present invention is, thehigher the ADCC activity of the antibody composition is. The antibodycomposition having higher ADCC activity includes an antibody compositionin which the ratio of sugar chains of the present invention ispreferably 20% or more, more preferably 30% or more, still morepreferably 40% or more, particularly preferably 50% or more, and mostpreferably 100%.

Furthermore, the present invention relates to a process for producing anantibody composition having higher ADCC activity than an antibodycomposition produced by its parent cell.

When the ratio of sugar chain of the present invention is higher thanthat of an antibody composition produced by its parent cell, theantibody composition has higher ADCC activity than the antibodycomposition produced by its parent cell.

The ADCC activity is a cytotoxic activity in which an antibody bound toa cell surface antigen on a tumor cell in the living body activates aneffector cell through the binding of the antibody Fc region and an Fcreceptor existing on the effector cell surface and thereby injuries thetumor cell and the like [Monoclonal Antibodies: Principles andApplications, Wiley-Liss, Inc., Chapter 2.1 (1995)]. The effector cellincludes a killer cell, a natural killer cell, an activated macrophageand the like.

The ratio of sugar chains in which fucose is not bound toN-acetylglucosamine in the reducing end in the sugar chains contained inthe composition which comprises an antibody molecule having complex typeN-glycoside-linked sugar chains in the Fc region can be determined byreleasing the sugar chains from the antibody molecule using a knownmethod such as hydrazinolysis or enzyme digestion [BiochemicalExperimentation Methods 23—Method for Studying Glycoprotein Sugar Chain(Japan Scientific Societies Press), edited by Reiko Takahashi (1989)],carrying out fluorescence labeling or radioisotope labeling of thereleased sugar chains and then separating the labeled sugar chains bychromatography. Also, the released sugar chains can also be determinedby analyzing it with the HPAED-PAD method [J. Liq. Chromatogr., 6, 1577(1983)].

The antibody molecule may be any antibody molecule, so long as itcomprises the Fc region of an antibody. Examples include an antibody, anantibody fragment, a fusion protein comprising an Fc region, and thelike.

Examples of the antibody include an antibody secreted by a hybridomacell prepared from a spleen cell of an animal immunized with an antigen;an antibody prepared by a genetic engineering technique, namely anantibody obtained by introducing an antibody gene-inserted antibodyexpression vector into a host cell; and the like. Specific examplesinclude an antibody produced by a hybridoma, a humanized antibody, ahuman antibody and the like.

A hybridoma is a cell which is obtained by cell fusion between a B cellobtained by immunizing a non-human mammal with an antigen and a myelomacell derived from a mouse, a rat or the like and which can produce amonoclonal antibody having the antigen specificity of interest.

The humanized antibody includes a human chimeric antibody, a humanCDR-grafted antibody and the like.

A human chimeric antibody is an antibody which comprises antibody VH andan antibody L chain V region (hereinafter referred to as “VL”), both ofa non-human animal antibody, a human antibody H chain C region(hereinafter also referred to as “CH”) and a human antibody L chain Cregion (hereinafter also referred to as “CL”). The non-human animal maybe any animal such as a mouse, a rat, a hamster or a rabbit, so long asa hybridoma can be prepared therefrom.

The human chimeric antibody can be produced by obtaining cDNAs encodingVH and VL from a monoclonal antibody-producing hybridoma, inserting theminto an expression vector for host cell comprising genes encoding humanantibody CH and human antibody CL to thereby construct a human chimericantibody expression vector, and then introducing the vector into a hostcell to express the antibody.

As the CH of human chimeric antibody, any CH can be used, so long as itbelongs to human immunoglobulin (hereinafter referred to as “hIg”) canbe used, and those belonging to the hIgG class are preferred, and anyone of the subclasses belonging to the hIgG class, such as hIgG1, hIgG2,hIgG3 and hIgG4, can be used. As the CL of human chimeric antibody, anyCL can be used, so long as it belongs to the hIg class, and thosebelonging to the κ class or λ class can be used.

A human CDR-grafted antibody is an antibody in which amino acidsequences of CDRs of VH and VL of a non-human animal antibody aregrafted into appropriate positions of VH and VL of a human antibody.

The human CDR-grafted antibody can be produced by constructing cDNAsencoding V regions in which the amino acid sequences of CDRs of VH andVL of a non-human animal antibody are grafted into the amino acidsequences of CDRs of VH and VL of a human antibody, inserting them intoan expression vector for host cell comprising genes encoding humanantibody CH and human antibody CL to thereby construct a humanCDR-grafted antibody expression vector, and then introducing theexpression vector into a host cell to express the human CDR-graftedantibody.

As the CH of human CDR-grafted antibody, any CH can be used, so long asit belongs to the hIg, and those of the hIgG class are preferred and anyone of the subclasses belonging to the hIgG class, such as hIgG1, hIgG2,hIgG3 and hIgG4, can be used. As the CL of human CDR-grafted antibody,any CL can be used, so long as it belongs to the hIg class, and thosebelonging to the κ class or λ class can be used.

A human antibody is originally an antibody naturally existing in thehuman body, but it also includes antibodies obtained from a humanantibody phage library, a human antibody-producing transgenicnon-transgenic animal or a human antibody-producing transgenic plant,which are prepared based on the recent advance in genetic engineering,cell engineering and embryological engineering techniques.

The antibody existing in the human body can be prepared, for example byisolating a human peripheral blood lymphocyte, immortalizing it by itsinfection with EB virus or the like and then cloning it to therebyobtain lymphocytes capable of producing the antibody, culturing thelymphocytes thus obtained, and purifying the antibody from the culture.

The human antibody phage library is a library in which antibodyfragments such as Fab and single chain antibody are expressed on thephage surface by inserting a gene encoding an antibody prepared from ahuman B cell into a phage gene. A phage expressing an antibody fragmenthaving the desired antigen binding activity can be recovered from thelibrary, using its activity to bind to an antigen-immobilized substrateas the marker. The antibody fragment can be converted further into ahuman antibody molecule comprising two full H chains and two full Lchains by genetic engineering techniques.

A human antibody-producing transgenic non-human animal is an animal inwhich a human antibody gene is introduced into cells. Specifically, ahuman antibody-producing transgenic non-human animal can be prepared byintroducing a human antibody gene into ES cell of a mouse, grafting theES cell into an early stage embryo of other mouse and then developingit. By introducing a human antibody gene into a fertilized egg anddeveloping it, the human antibody-producing transgenic non-human animalcan be also prepared. A human antibody is prepared from the humanantibody-producing transgenic non-human animal by obtaining a humanantibody-producing hybridoma by a hybridoma preparation method usuallycarried out in non-human mammals, culturing the obtained hybridoma andaccumulating the human antibody in the culture.

The transgenic non-human animal includes cattle, sheep, goat, pig,horse, mouse, rat, fowl, monkey, rabbit and the like.

In the present invention, as the antibody, preferred are an antibodywhich recognizes a tumor-related antigen, an antibody which recognizesan allergy- or inflammation-related antigen, an antibody whichrecognizes cardiovascular disease-related antigen, an antibody whichrecognizes an autoimmune disease-related antigen or an antibody whichrecognizes a viral or bacterial infection-related antigen, and a humanantibody which belongs to the IgG class is preferred.

An antibody fragment is a fragment which comprises the Fc region of anantibody. The antibody fragment may be any fragment, so long as itcomprise the Fc region of the above-described antibody. The antibodyfragment includes an H chain monomer, an H chain dimmer and the like.

A fusion protein comprising the Fc region is a protein which is obtainedby fusing an antibody comprising the Fc region of an antibody or theantibody fragment with a protein such as an enzyme or a cytokine.

The present invention is explained below in detail.

1. Preparation of Cell of the Present Invention

(1) Preparation of Cell into which an RNA Capable of Suppressing theFunction of a GDP-Mannose Converting Enzyme

The cell into which an RNA capable of suppressing the function of aGDP-mannose converting enzyme in the present invention can be prepared,for example, as follows.

A cDNA or a genomic DNA of a GDP-mannose converting enzyme is prepared.

The nucleotide sequence of the prepared cDNA or genomic DNA isdetermined.

Based on the determined DNA sequence, a construct of an RNAi genecomprising a coding region or a non-coding region of the GDP-mannoseconverting enzyme at an appropriate length is designed.

In order to express the RNAi gene in a cell, a recombinant vector isprepared by inserting a fragment or full length of the prepared DNA intodownstream of the promoter of an appropriate expression vector.

A transformant is obtained by introducing the recombinant vector into ahost cell suitable for the expression vector.

The cell of the present invention can be obtained by selecting atransformant based on the activity of the introduced GDP-mannoseconverting enzyme or the sugar chain structure of the produced antibodymolecule or the glycoprotein on the cell surface.

As the host cell, any cell such as an yeast, an animal cell, an insectcell or a plant cell can be used, so long as it has a gene of the targetGDP-mannose converting enzyme. Examples include host cells described inthe following item 2.

As the expression vector, a vector which is autonomously replicable inthe host cell or can be integrated into the chromosome and comprises apromoter at such a position that the designed RNAi gene can betranscribed is used. Examples include the expression vectors transcribedby polymerase III or the expression vectors described in the followingitem 2.

As the method for introducing a gene into various host cells, themethods for introducing recombinant vectors suitable for various hostcells described in the following item 2 can be used.

As a method for obtaining a cDNA or genomic DNA of the GDP-mannoseconverting enzyme, the following method is exemplified.

Preparation Method of cDNA:

A total RNA or mRNA is prepared from various host cells.

A cDNA library is prepared from the prepared total RNA or mRNA.

Degenerative primers are produced based on a known amino acid sequenceof the GDP-mannose converting enzyme, such as an amino acid sequence ofthe enzyme in human, and a gene fragment encoding the GDP-mannoseconverting enzyme is obtained by PCR using the prepared cDNA library asthe template.

A cDNA of the GDP-mannose converting enzyme can be obtained by screeningthe cDNA library using the obtained gene fragment as a probe.

The mRNA of various host cells may be a commercially available product(e.g., manufactured by Clontech) or may be prepared from various hostcells as follows. The method for preparing a total mRNA from varioushost cells include the guanidine thiocyanate-cesium trifluoroacetatemethod [Methods in Enzymology, 154, 3 (1987)], the acidic guanidinethiocyanate phenol chloroform (AGPC) method [Analytical Biochemistry,162, 156 (1987); Experimental Medicine (Jikken Igaku), 9, 1937 (1991)]and the like.

Furthermore, a method for preparing mRNA as poly(A)⁺ RNA from a totalRNA includes the oligo(dT)-immobilized cellulose column method(Molecular Cloning, Second Edition).

In addition, mRNA can be prepared using a kit such as Fast Track mRNAIsolation Kit (manufactured by Invitrogen) or Quick Prep mRNAPurification Kit (manufactured by Pharmacia).

A cDNA library is prepared from the prepared mRNA of various host cells.The method for preparing cDNA libraries includes the methods describedin Molecular Cloning, Second Edition; Current Protocols in MolecularBiology, A Laboratory Manual, 2nd Ed. (1989); and the like, or methodsusing commercially available kits such as SuperScript Plasmid System forcDNA Synthesis and Plasmid Cloning (manufactured by Life Technologies)and ZAP-cDNA Synthesis Kit (manufactured by STRATAGENE).

As the cloning vector for preparing the cDNA library, any vector such asa phage vector or a plasmid vector can be used, so long as it isautonomously replicable in Escherichia coli K12. Examples include ZAPExpress [manufactured by STRATAGENE, Strategies, 5, 58 (1992)],pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989)], LambdaZAP II (manufactured by STRATAGENE), λgt10 and λgt11 [DNA Cloning, APractical Approach, 1, 49 (1985)], λTriplEx (manufactured by Clontech),λExCell (manufactured by Pharmacia), pT7T318U (manufactured byPharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)], pUC18 [Gene, 33, 103(1985)] and the like.

Any microorganism can be used as the host microorganism for preparingthe cDNA library, and Escherichia coli is preferably used. Examplesinclude Escherichia coli XL1-Blue MRF′ [manufactured by STRATAGENE,Strategies, 5, 81 (1992)], Escherichia coli C600 [Genetics, 39, 440(1954)], Escherichia coli Y1088 [Science, 222, 778 (1983)], Escherichiacoli Y1090 [Science, 222, 778 (1983)], Escherichia coli NM522 [J. Mol.Biol., 166, 1 (1983)], Escherichia coli K802 [J. Mol. Biol., 16, 118(1966)], Escherichia coli JM105 [Gene, 38, 275 (1985)] and the like

The cDNA library can be used as such in the subsequent analysis, but inorder to obtain a full length cDNA as efficient as possible bydecreasing the ratio of an infull length cDNA, a cDNA library preparedaccording to the oligo cap method developed by Sugano et al. [Gene, 138,171 (1994); Gene, 200, 149 (1997); Protein, Nucleic Acid, Koso(Tanpakushitu, Kakusan, Koso), 41, 603 (1996); Experimental Medicine(Jikken Igaku), 11, 2491 (1993); cDNA Cloning (Yodo-sha) (1996); Methodsfor Preparing Gene Libraries (Yodo-sha) (1994)] can be used in thefollowing analysis.

Based on the amino acid sequence of the GDP-mannose converting enzyme,degenerative primers specific for the 5′-terminal and 3′-terminalnucleotide sequences of a nucleotide sequence presumed to encode theamino acid sequence are prepared, and DNA is amplified by PCR [PCRProtocols, Academic Press (1990)] using the prepared cDNA library as thetemplate to obtain a gene fragment encoding the GDP-mannose convertingenzyme.

It can be confirmed that the obtained gene fragment is a DNA encodingthe GDP-mannose converting enzyme by a method generally used foranalyzing a nucleotide such as the dideoxy method of Sanger et al.[Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] or a nucleotide sequenceanalyzer such as ABIPRISM 377 DNA Sequencer (manufactured by PEBiosystems).

A cDNA encoding the GDP-mannose converting enzyme can be obtained bycarrying out colony hybridization or plaque hybridization (MolecularCloning, Second Edition) for the cDNA or cDNA library synthesized fromthe mRNA contained in various host cells, using the gene fragment as aDNA probe.

Also, a cDNA encoding the GDP-mannose converting enzyme can also beobtained by carrying out screening by PCR using the cDNA or cDNA librarysynthesized from the mRNA contained in various host cells as thetemplate and using the primers used for obtaining the gene fragmentencoding the GDP-mannose converting enzyme.

The nucleotide sequence of the obtained DNA encoding the GDP-mannoseconverting enzyme is analyzed from its terminus and determined by amethod generally used for analyzing a nucleotide such as the dideoxymethod of Sanger et al. [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] ora nucleotide sequence analyzer such as ABI PRISM 377 DNA Sequencer(manufactured by PE Biosystems).

A gene encoding the GDP-mannose converting enzyme can also be determinedfrom genes in databases by searching nucleotide sequence databases suchas GenBank, EMBL and DDBJ using a homology retrieving program such asBLAST based on the determined cDNA nucleotide sequence.

The nucleotide sequence of a gene encoding the GDP-mannose convertingenzyme obtained by the above method includes the nucleotide sequencerepresented by any one of SEQ ID NOs:8 to 10.

The cDNA of the GDP-mannose converting enzyme can also be obtained bychemically synthesizing it with a DNA synthesizer such as DNASynthesizer model 392 manufactured by Perkin Elmer using thephosphoamidite method, based on the determined DNA nucleotide sequence.

As an example of the method for preparing a genomic DNA of theGDP-mannose converting enzyme, the method described below isexemplified.

Preparation Method of Genomic DNA:

The method for preparing genomic DNA includes known methods described inMolecular Cloning, Second Edition; Current Protocols in MolecularBiology; and the like. In addition, a genomic DNA of the GDP-mannoseconverting enzyme can also be isolated using Genome DNA LibraryScreening System (manufactured by Genome Systems) UniversalGenomeWalker™ Kits (manufactured by CLONTECH), or the like.

The following method can be exemplified as the method for selecting atransformant based on the activity of the GDP-mannose converting enzyme.

Method for Selecting Transformant:

The method for selecting a cell in which the activity of the GDP-mannoseconverting enzyme is decreased includes biochemical methods or geneticengineering techniques described in New Biochemical ExperimentationSeries 3—Saccharides I, Glycoprotein (Tokyo Kagaku Dojin), edited byJapanese Biochemical Society (1988); Cell Engineering, Supplement,Experimental Protocol Series, Glycobiology Experimental Protocol,Glycoprotein, Glycolipid and Proteoglycan (Shujun-sha), edited byNaoyuki Taniguchi, Akemi Suzuki, Kiyoshi Furukawa and Kazuyuki Sugawara(1996); Molecular Cloning, Second Edition; Current Protocols inMolecular Biology; and the like. The biochemical method includes amethod in which the enzyme activity is evaluated using anenzyme-specific substrate. The genetic engineering techniques includethe Northern analysis, RT-PCR and the like which measures the amount ofmRNA of a gene encoding the GDP-mannose converting enzyme.

Furthermore, the method for selecting a cell based on morphologicalchange caused by decrease of the activity of the GDP-mannose convertingenzyme includes a method for selecting a transformant based on the sugarchain structure of a produced glycoprotein molecule, a method forselecting a transformant based on the sugar chain structure of aglycoprotein on a cell surface, and the like. The method for selecting atransformant using the sugar chain structure of a glycoprotein-producingmolecule includes method described in the item 5 below. The method forselecting a transformant using the sugar chain structure of aglycoprotein on a cell surface includes a method in which a cloneresistant to a lectin which recognizes a sugar chain structure wherein1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in the N-glycoside-linked sugar chain isselected. Examples include a method using a lectin described in SomaticCell Mol. Genet., 12, 51 (1986).

As the lectin, any lectin can be used, so long as it is a lectin whichrecognizes a sugar chain structure in which 1-position of fucose isbound to 6-position of N-acetylglucosamine in the reducing end throughα-bond in the N-glycoside-linked sugar chain. Examples include a Lensculinaris agglutinin LCA (lentil agglutinin derived from Lensculinaris), a Pisum sativum agglutinin PSA (pea lectin derived fromPisum sativum), a Vicia faba agglutinin VFA (agglutinin derived fromVicia faba), an Aleuria aurantia lectin AAL (lectin derived from Aleuriaaurantia) and the like.

Specifically, the cell of the present invention can be selected byculturing cells for 1 day to 2 weeks, preferably from 3 days to 1 week,in a medium containing the above lectin at a concentration of 10 μg/mlto 10 mg/ml, preferably 0.5 to 2 mg/ml, subculturing surviving cells orpicking up a colony and transferring it into a culture vessel, andsubsequently continuing the culturing in the lectin-containing medium.

The RNAi gene for suppressing the mRNA amount of a gene encoding theGDP-mannose converting enzyme can be prepared in the usual method or byusing a DNA synthesizer.

The construct of the RNAi gene can be designed according to thedescription in Nature, 391, 806 (1998); Proc. Natl. Acad. Sci. USA, 95,15502 (1998); Nature, 395, 854 (1998); Proc. Natl. Acad. Sci. USA, 96,5049 (1999); Cell, 95, 1017 (1998); Proc. Natl. Acad. Sci. USA, 96, 1451(1999); Proc. Natl. Acad. Sci. USA, 95, 13959 (1998); Nature Cell Biol.,2, 70 (2000) and the like.

In addition, the cell of the present invention can also be obtainedwithout using an expression vector, by directly introducing adouble-stranded RNA which is designed based on the nucleotide sequenceof the GDP-mannose converting enzyme into a host cell.

The double-stranded RNA can be prepared in the usual method or by usinga DNA synthesizer. Specifically, it can be prepared based on thesequence information of an oligonucleotide having a correspondingsequence of 10 to 40 bases, preferably 10 to 30 bases, and morepreferably 15 to 30 bases, among complementary RNA nucleotide sequencesof a cDNA and a genomic DNA of a GDP-mannose converting enzyme bysynthesizing an oligonucleotide which corresponds to a sequencecomplementary to the oligonucleotide (antisense oligonucleotide). Theoligonucleotide and the antisense oligonucleotide may be independentlysynthesized or may be linked via a spacer nucleotide which does notobstruct the formation of the double-stranded RNA.

The oligonucleotide includes an oligo RNA and derivatives of theoligonucleotide (hereinafter referred to as “oligonucleotidederivatives”).

The oligonucleotide derivatives includes oligonucleotide derivatives inwhich a phosphodiester bond in the oligonucleotide is converted into aphosphorothioate bond, an oligonucleotide derivative in which aphosphodiester bond in the oligonucleotide is converted into an N3′-P5′phosphoamidate bond, an oligonucleotide derivative in which ribose and aphosphodiester bond in the oligonucleotide are converted into apeptide-nucleic acid bond, an oligonucleotide derivative in which uracilin the oligonucleotide is substituted with C-5 propynyluracil, anoligonucleotide derivative in which uracil in the oligonucleotide issubstituted with C-5 thiazoleuracil, an oligonucleotide derivative inwhich cytosine in the oligonucleotide is substituted with C-5propynylcytosine, an oligonucleotide derivative in which cytosine in theoligonucleotide is substituted with phenoxazine-modified cytosine, anoligonucleotide derivative in which ribose in the oligonucleotide issubstituted with 2′-O-propylribose and an oligonucleotide derivative inwhich ribose in the oligonucleotide is substituted with2′-methoxyethoxyribose [Cell Technology (Saibo Kogaku), 16, 1463(1997)].

(2) Preparation of Cell into which an RNA Capable of Suppressing theFunction of an α1,6-Fucose Modifying Enzyme and an RNA Capable ofSuppressing the Function of a GDP-Fucose Synthase or an RNA Capable ofSuppressing the Function of a Protein Relating to Transport of anIntracellular Sugar Nucleotide, GDP-Fucose, to the Golgi Body areIntroduced

A process for producing the cell into which an RNA capable ofsuppressing the function of an α1,6-fucose modifying enzyme and an RNAcapable of suppressing the function of a GDP-fucose synthase or an RNAcapable of suppressing the function of a protein relating to transportof an intracellular sugar nucleotide, GDP-fucose, to the Golgi body areintroduced is explained below based on, as an example, a cell into whichan RNA capable of suppressing the function of an α1,6-fucose modifyingenzyme and an RNA capable of suppressing the function of a GDP-fucosesynthase are introduced. The cell into which an RNA capable ofsuppressing the function of an α1,6-fucose modifying enzyme and an RNAcapable of suppressing the function of a protein relating to transportof an intracellular sugar nucleotide, GDP-fucose, to the Golgi body areintroduced can be prepared similarly.

A cDNA or a genomic DNA of each of an α1,6-fucose modifying enzyme and aGDP-fucose synthase is prepared.

The nucleotide sequence of the prepared cDNA or genomic DNA isdetermined.

Based on the determined DNA sequence, a construct of an RNAi genecomprising a coding region or a non-coding region of the α1,6-fucosemodifying enzyme and the GDP-fucose synthase at an appropriate length isdesigned.

In order to express the RNAi gene in a cell, a recombinant vector isprepared by inserting a fragment or full length of the prepared DNA intodownstream of the promoter of an appropriate expression vector.

A transformant is obtained by introducing the recombinant vector into ahost cell suitable for the expression vector.

The cell of the present invention can be obtained by selecting atransformant based on the activity of the introduced α1,6-fucosemodifying enzyme or GDP-fucose synthase or the sugar chain structure ofthe produced antibody molecule or the glycoprotein on the cell surface.

As the host cell, any cell such as an yeast, an animal cell, an insectcell or a plant cell can be used, so long as it has the target genes ofthe α1,6-fucose modifying enzyme and the GDP-fucose synthase. Examplesinclude cells described in the following item 2.

As the expression vector, a vector which is autonomously replicable inthe above host cell or can be integrated into the chromosome andcomprises a promoter at such a position that the designed RNAi gene canbe transcribed is used. Examples include the expression vectortranscribed by polemerase III or the expression vectors described in thefollowing item 2.

As the method for introducing a gene into various host cells, themethods for introducing recombinant vectors suitable for various hostcells described in the following item 2 can be used.

A cDNA and a genomic DNA of the α1,6-fucose modifying enzyme or theGDP-fucose synthase can be obtained, for example, in the same manner asthe method described in (1).

As a method for selecting a transformant based on the activity of theα1,6-fucose modifying enzyme or the GDP-fucose synthase, the followingmethod is exemplified.

Method for Selecting Transformant

The method for selecting a cell in which the activity of the α1,6-fucosemodifying enzyme or the GDP-fucose synthase is decreased includes thebiochemical method and genetic engineering method described in the above(1).

Also, the method for selecting a cell based on morphological changecaused by a result in which the activity of α1,6-fucose modifying enzymeor the GDP-fucose synthase is decreased include the method described inthe above (1).

The RNAi gene for suppressing the amount of the mRNA of the α1,6-fucosemodifying enzyme gene or the GDP-fucose synthase gene can be prepared inthe usual method or by using a DNA synthesizer.

A construct of the RNAi can be designed in the same manner as in themethod described in the above (1).

In addition, the cell of the present invention can also be obtainedwithout using an expression vector, by directly introducing adouble-stranded RNA which is designed based on the nucleotide sequenceof the α1,6-fucose modifying enzyme and a double-stranded RNA which isdesigned based on the nucleotide sequence of the GDP-fucose synthaseinto a host cell.

The double-stranded RNA can be prepared in the same manner as in themethod described in the above (1).

2. Method for Producing Glycoprotein Referring to Antibody Compositionas an Example.

A method for producing a glycoprotein using the cell of the presentinvention is explained below by referring to an antibody composition asan example.

The antibody composition can be expressed in a cell of the presentinvention and obtained by the method described in Molecular Cloning,Second Edition; Current Protocols in Molecular Biology; Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, 1988 (hereinafterreferred to as “Antibodies”); Monoclonal Antibodies: Principles andPractice, Third Edition, Acad. Press, 1993 (hereinafter referred to as“Monoclonal Antibodies”); or Antibody Engineering, A Practical Approach,IRL Press at Oxford University Press (hereinafter referred to as“Antibody Engineering”), for example, as follows.

A cDNA encoding an antibody molecule is prepared.

Based on the prepared full length cDNA of antibody molecule, a DNAfragment of an appropriate length comprising a coding region of theprotein is prepared, if necessary.

A recombinant vector is prepared by inserting the DNA fragment or thefull length cDNA into downstream of the promoter of an appropriateexpression vector.

A transformant producing the antibody molecule can be obtained byintroducing the recombinant vector into a host cell suitable for theexpression vector.

In the present invention, as the host cell for producing the antibodycomposition, any cell such as a yeast, an animal cell, an insect cell ora plant cell can be used, so long as it is the cell of the presentinvention prepared in the above item 1 and can express the geneinterest. An animal cell is preferred.

A cell such as an yeast, an animal cell, an insect cell or a plant cellinto which an enzyme relating to the modification of anN-glycoside-linked sugar chain which binds to the Fc region of theantibody molecule is introduced by a genetic engineering technique canalso be used as the host cell.

As the expression vector, a vector which is autonomously replicable inthe above host cell or can be integrated into the chromosome andcomprises a promoter at such a position that the DNA encoding theantibody molecule of interest can be transcribed is used.

The cDNA can be prepared from a human or non-human animal tissue or cellby using a probe primer specific for the antibody molecule of interestaccording to “Preparation method of cDNA” described in the above item 1.

When an yeast is used as the host cell, the expression vector includesYEP13 (ATCC 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419) and the like.

Any promoter can be used, so long as it can function in an yeast.Examples include a promoter of a gene of the glycolytic pathway such asa hexose kinase, PHO5 promoter, PGK promoter, GAP promoter, ADHpromoter, gal 1 promoter, gal 10 promoter, heat shock protein promoter,MF α1 promoter, CUP 1 promoter and the like. The host cell includesyeasts belonging to the genus Saccharomyces, the genusSchizosaccharomyces, the genus Kluyveromyces, the genus Trichosporon,the genus Schwanniomyces and the like, such as Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulansand Schwanniomyces alluvius.

As the method for introducing the recombinant vector, any method can beused, so long as it can introduce DNA into yeast. Examples includeelectroporation [Methods in Enzymology, 194, 182 (1990)], thespheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], thelithium acetate method [J. Bacteriol., 153, 163 (1983)], the methoddescribed in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like.

When an animal cell is used as the host cell, the expression vectorincludes pcDNAI, pcDM8 (available from Funakoshi), pAGE107 [JapanesePublished Unexamined Patent Application No. 22979/91; Cytotechnology, 3,133 (1990)], pAS3-3 (Japanese Published Unexamined Patent ApplicationNo. 227075/90), pCDM8 [Nature, 329, 840 (1987)], pcDNAI/Amp(manufactured by Invitrogen), pREP4 (manufactured by Invitrogen),pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210 and the like.

Any promoter can be used, so long as it can function in an animal cell.Examples include a promoter of IE (immediate early) gene ofcytomegalovirus (CMV), an early promoter of SV40, a promoter ofretrovirus, a promoter of metallothionein, a heat shock promoter, an SRαpromoter and the like. Also, an enhancer of the IE gene of human CMV canbe used together with the promoter.

The host cell includes a human cell such as Namalwa cell, NM-F9 cell andPER.C6 cell, a monkey cell such as COS cell, a Chinese hamster cell suchas CHO cell and HBT5637 (Japanese Published Unexamined PatentApplication No. 299/88), a rat myeloma cell, a mouse myeloma cell, acell derived from syrian hamster kidney, an embryonic stem cell, afertilized egg cell and the like.

As the method for introducing the recombinant vector, any method can beused, so long as it can introduce a DNA into an animal cell. Examplesinclude electroporation [Cytotechnology, 3, 133 (1990)], the calciumphosphate method (Japanese Published Unexamined Patent Application No.227075/90), the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413(1987)], the injection method [Manipulating the Mouse Embryo, ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press(1994), hereinafter also referred to as “Manipulating the Mouse Embryo,Second Edition”], a method using a particle gun (gene gun) (JapanesePatent No. 2606856, Japanese Patent No. 2517813), the DEAE-dextranmethod [Biomanual Series 4—Gene Transfer and Expression Analysis(Yodo-sha), edited by Takashi Yokota and Kenichi Arai (1994)], the virusvector method [Manipulating Mouse Embryo, Second Edition] and the like.

When an insect cell is used as the host cell, the protein can beexpressed by the method described in Current Protocols in MolecularBiology, Baculovirus Expression Vectors, A Laboratory Manual, W.H.Freeman and Company, New York (1992), Bio/Technology, 6, 47 (1988) orthe like.

That is, the protein can be expressed by co-introducing a recombinantgene-introducing vector and a baculovirus into an insect cell to obtaina recombinant virus in an insect cell culture supernatant and theninfecting the insect cell with the recombinant virus.

The gene-introducing vector used in the method includes pVL1392,pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like.

The baculovirus includes Autographa californica nuclear polyhedrosisvirus which is infected by an insect of the family Barathra.

The insect cell includes Spodoptera frugiperda oocytes Sf9 and Sf21[Current Protocols in Molecular Biology, Baculovirus Expression Vectors,A Laboratory Manual, W.H. Freeman and Company, New York (1992)], aTrichoplusia ni oocyte High 5 (manufactured by Invitrogen) and the like.

The method for the co-introducing the above-mentioned recombinantgene-introducing vector and the above-mentioned baculovirus forpreparing the recombinant virus to an insect cell includes the calciumphosphate method (Japanese Published Unexamined Patent Application No.227075/90), the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413(1987)] and the like.

When a plant cell is used as the host cell, the expression vectorincludes Ti plasmid, tobacco mosaic virus vector and the like.

As the promoter, any promoter can be used, so long as it can function ina plant cell. Examples include cauliflower mosaic virus (CaMV) 35Spromoter, rice actin 1 promoter and the like.

The host cell includes plant cells of tobacco, potato, tomato, carrot,soybean, rape, alfalfa, rice, wheat, barley, and the like.

As the method for introducing the recombinant vector, any method can beused, so long as it can introduce a DNA into a plant cell. Examplesinclude a method using Agrobacterium (Japanese Published UnexaminedPatent Application No. 140885/84, Japanese Published Unexamined PatentApplication No. 70080/85, WO 94/00977), electroporation (JapanesePublished Unexamined Patent Application No. 251887/85), a method using aparticle gun (gene gun) (Japanese Patent No. 2606856, Japanese PatentNo. 2517813) and the like.

As the method for expressing an antibody gene, secretion production,expression of a fusion protein and the like can be carried out inaccordance with the method described in Molecular Cloning, SecondEdition or the like, in addition to the direct expression.

The antibody composition can be produced by culturing the thus obtainedtransformant in a medium to form and accumulate the antibody molecule inthe culture and recovering the antibody composition from the culture.The method for culturing the transformant can be carried out by aconventional method used for the culturing of a host cell.

As the medium for culturing a transformant obtained using a eukaryote,such as an yeast, as the host cell, the medium may be either a naturalmedium or a synthetic medium, so long as it comprises materials such asa carbon source, a nitrogen source, an inorganic salt and the like whichcan be assimilated by the organism and culturing of the transformant canbe efficiently carried out.

As the carbon source, those which can be assimilated by the organism canbe used. Examples include carbohydrates such as glucose, fructose,sucrose, molasses containing them, starch and starch hydrolysate;organic acids such as acetic acid and propionic acid; alcohols such asethanol and propanol; and the like.

The nitrogen source includes ammonia; ammonium salts of inorganic acidor organic acid such as ammonium chloride, ammonium sulfate, ammoniumacetate and ammonium phosphate; other nitrogen-containing compounds;peptone; meat extract; yeast extract; corn steep liquor; caseinhydrolysate; soybean meal; soybean meal hydrolysate; various fermentedcells and hydrolysates thereof; and the like.

The inorganic salt includes potassium dihydrogen phosphate, dipotassiumhydrogen phosphate, magnesium phosphate, magnesium sulfate, sodiumchloride, ferrous sulfate, manganese sulfate, copper sulfate, calciumcarbonate, and the like.

The culture is carried out generally under aerobic conditions such asshaking culture or submerged-aeration stirring culture. The culturingtemperature is preferably 15 to 40° C., and the culturing time isgenerally 16 hours to 7 days. In the culture, the pH is maintained at 3to 9. The pH is adjusted using an inorganic or organic acid, an alkalisolution, urea, calcium carbonate, ammonia or the like.

If necessary, an antibiotic such as ampicillin or tetracycline can beadded to the medium in the culture.

When an yeast transformed with a recombinant vector obtained using aninducible promoter as the promoter is cultured, an inducer can be addedto the medium, if necessary. For example, when an yeast transformed witha recombinant vector obtained using lac promoter is cultured,isopropyl-β-D-thiogalactopyranoside can be added to the medium, and whenan yeast transformed with a recombinant vector obtained using trppromoter is cultured, indoleacrylic acid can be added to the medium.

When a transformant obtained using an animal cell as the host cell iscultured, the medium includes generally used RPMI 1640 medium [TheJournal of the American Medical Association, 199, 519 (1967)], Eagle'sMEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium[Virology, 8, 396 (1959)], 199 medium [Proceeding of the Society for theBiological Medicine, 73, 1 (1950)] and Whitten's medium [DevelopmentalEngineering Experimentation Manual—Preparation of Transgenic Mice(Kodan-sha), edited by Motoya Katsuki (1987)], the medium obtained byadding fetal bovine serum, etc. to these medium, and the like.

The culture is carried out generally at a pH of 6 to 8 and 30 to 40° C.for 1 to 7 days in the presence of 5% CO₂. Culturing can also be carriedout by culturing using a method such as fed-batch culture orhollow-fiber culture for 1 day to several months.

If necessary, an antibiotic such as kanamycin or penicillin can be addedto the medium in the culture.

The medium for use in the culture of a transformant obtained using aninsect cell as the host cell includes generally used TNM-FH medium(manufactured by Pharmingen), Sf-900 II SFM medium (manufactured by LifeTechnologies), ExCell 400 and ExCell 405 (both manufactured by JRHBiosciences), Grace's Insect Medium [Nature, 195, 788 (1962)] and thelike.

The culture is carried out generally at a pH of 6 to 7 and 25 to 30° C.for 1 to 5 days.

In addition, antibiotics such as gentamicin can be added to the mediumin the culture, if necessary.

A transformant obtained using a plant cell as the host cell can becultured as a cell or after differentiating it into a plant cell ororgan. The medium for culturing the transformant includes generally usedMurashige and Skoog (MS) medium and White medium, the medium obtained byadding a plant hormone such as auxin or cytokinin to these medium, andthe like.

The culture is carried out generally at a pH of 5 to 9 and 20 to 40° C.for 3 to 60 days.

Also, an antibiotic such as kanamycin or hygromycin can be added to themedium in the culture, if necessary.

Thus, an antibody composition can be produced by culturing atransformant derived from yeast, an animal cell or a plant cell whichcomprises a recombinant vector into which a DNA encoding an antibodymolecule is inserted, in accordance with a general culturing method, tothereby produce and accumulate the antibody composition, and thenrecovering the antibody composition from the culture.

The process for producing an antibody composition includes a method ofintracellular expression in a host cell, a method of extracellularsecretion from a host cell, and a method of production on a host cellmembrane outer envelope. The method can be selected by changing the hostcell used or the structure of an antibody molecule produced.

When the antibody composition is produced in a host cell or on an outermembrane of a host cell, it can be positively secreted extracellularlyin accordance with the method of Paulson et al. [J. Biol. Chem., 264,17619 (1989)], the method of Lowe et al. [Proc. Natl. Acad. Sci. USA,86, 8227 (1989), Genes Develop., 4, 1288 (1990)], the methods describedin Japanese Published Unexamined Patent Application No. 336963/93 andJapanese Published Unexamined Patent Application No. 823021/94 and thelike.

That is, an antibody molecule of interest can be positively secretedextracellularly from a host cell by inserting a DNA encoding theantibody molecule and a DNA encoding a signal peptide suitable for theexpression of the antibody molecule into an expression vector using agene recombination technique, and introducing the expression vector intothe host cell to express the antibody molecule.

Also, the production can be increased in accordance with the methoddescribed in Japanese Published Unexamined Patent Application No.227075/90 using a gene amplification system using a dihydrofolatereductase gene.

In addition, the antibody composition can also be produced using agene-introduced animal individual (transgenic non-human animal) or aplant individual (transgenic plant) which is constructed by theredifferentiation of an animal or plant cell into which the gene isintroduced.

When the transformant is an animal individual or a plant individual, anantibody composition can be produced in accordance with a general methodby rearing or cultivating it to thereby produce and accumulate theantibody composition and then recovering the antibody composition fromthe animal or plant individual.

The process for producing an antibody composition using an animalindividual includes a method in which the antibody composition ofinterest is produced in an animal constructed by introducing a gene inaccordance with a known method [American Journal of Clinical Nutrition,63, 639S (1996); American Journal of Clinical Nutrition, 63, 627S(1996); Bio/Technology, 9, 830 (1991)].

In the case of an animal individual, an antibody composition can beproduced by rearing a transgenic non-human animal into which a DNAencoding an antibody molecule is introduced to thereby produce andaccumulate the antibody composition in the animal, and then recoveringthe antibody composition from the animal. The place in the animal wherethe composition is produced and accumulated includes milk (JapanesePublished Unexamined Patent Application No. 309192/88) and eggs of theanimal. As the promoter used in this case, any promoter can be used, solong as it can function in an animal. Preferred examples include mammarygland cell-specific promoters such as α casein promoter, β caseinpromoter, β lactoglobulin promoter, whey acidic protein promoter and thelike.

The process for producing an antibody composition using a plantindividual includes a method in which an antibody composition isproduced by cultivating a transgenic plant into which a DNA encoding anantibody molecule is introduced by a known method [Tissue Culture(Soshiki Baiyo), 20 (1994); Tissue Culture (Soshiki Baiyo), 21 (1995);Trends in Biotechnology, 15, 45 (1997)] to produce and accumulate theantibody composition in the plant, and then recovering the antibodycomposition from the plant.

Regarding purification of an antibody composition produced by atransformant into which a gene encoding an antibody molecule isintroduced, for example, when the antibody composition isintracellularly expressed in a dissolved state, the cells afterculturing are recovered by centrifugation, suspended in an aqueousbuffer and then disrupted using ultrasonicator, French press, MantonGaulin homogenizer, dynomill or the like to obtain a cell-free extract,which is centrifuged to obtain a supernatant, and a purified preparationof the antibody composition can be obtained by subjecting thesupernatant to a general enzyme isolation and purification techniquessuch as solvent extraction; salting out with ammonium sulfate etc.;desalting; precipitation with an organic solvent; anion exchangechromatography using a resin such as diethylaminoethyl (DEAE)-sepharose,DIAION HPA-75 (manufactured by Mitsubishi Chemical); cation exchangechromatography using a resin such as S-Sepharose FF (manufactured byPharmacia); hydrophobic chromatography using a resin such asbutyl-Sepharose or phenyl-Sepharose; gel filtration using a molecularsieve; affinity chromatography; chromatofocusing; electrophoresis suchas isoelectric focusing; and the like which may be used alone or incombination.

When the antibody composition is expressed intracellularly by forming aninclusion body, the cells are recovered, disrupted and centrifuged inthe same manner, and the inclusion body of the antibody composition isrecovered as a precipitation fraction. The recovered inclusion body ofthe antibody composition is solubilized with a protein denaturing agent.The antibody composition is made into a normal three-dimensionalstructure by diluting or dialyzing the solubilized solution, and then apurified preparation of the antibody composition is obtained by the sameisolation purification method as above.

When the antibody composition is secreted extracellularly, the antibodycomposition can be recovered from the culture supernatant. That is, theculture is treated by a technique such as centrifugation in the samemanner as above to obtain a soluble fraction, and a purified preparationof the antibody composition can be obtained from the soluble fraction bythe same isolation purification method as above.

The antibody composition thus obtained includes an antibody, thefragment of the antibody, a fusion protein comprising the Fc region ofthe antibody, and the like.

As examples for obtaining the antibody composition, processes forproducing a composition of humanized antibody composition and Fc fusionprotein are described below in detail, but other antibody compositionscan also be obtained in accordance with the methods mentioned above andthe said method.

A. Preparation of Humanized Antibody Composition

(1) Construction of Vector for Expression of Humanized Antibody

A vector for expression of humanized antibody is an expression vectorfor animal cell into which genes encoding CH and CL of a human antibodyare inserted, which can be constructed by cloning each of genes encodingCH and CL of a human antibody into an expression vector for animal cell.

The C regions of a human antibody may be CH or CL of any human antibody.Examples include the C region belonging to IgG1 subclass in the H chainof a human antibody (hereinafter referred to as “hCγ1”), the C regionbelonging to κ class in the L chain of a human antibody (hereinafterreferred to as “hCκ”), and the like.

As the genes encoding CH and CL of a human antibody, a chromosomal DNAcomprising an exon and an intron can be used, and a cDNA can also beused.

As the expression vector for animal cell, any vector can be used, solong as a gene encoding the C region of a human antibody can be insertedthereinto and expressed therein. Examples include pAGE107[Cytotechnology, 3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307(1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci.USA, 78, 1527 (1981), pSG1 β d2-4 [Cytotechnology, 4, 173 (1990)] andthe like. The promoter and enhancer in the expression vector for animalcell include SV40 early promoter and enhancer [J. Biochem., 101, 1307(1987)], Moloney mouse leukemia virus LTR [Biochem. Biophys. Res.Commun., 149, 960 (1987)], immunoglobulin H chain promoter [Cell, 41,479 (1985)] and enhancer [Cell, 33, 717 (1983)], and the like.

The vector for expression of humanized antibody may be either of a typein which genes encoding the H chain and L chain of an antibody exist onseparate vectors or of a type in which both genes exist on the samevector (hereinafter referred to as “tandem type”). In respect ofeasiness of construction of a vector for expression of humanizedantibody, easiness of introduction into animal cells, and balancebetween the expression amounts of the H and L chains of an antibody inanimal cells, a tandem type of the vector for expression of humanizedantibody is more preferred [J. Immunol. Methods, 167, 271 (1994)].

The constructed vector for expression of humanized antibody can be usedfor expression of a human chimeric antibody and a human CDR-graftedantibody in animal cells.

(2) Obtaining of cDNA Encoding V Region of Non-Human Animal Antibody

cDNAs encoding VH and VL of a non-human animal antibody such as a mouseantibody can be obtained in the following manner.

A cDNA is synthesized from mRNA extracted from a hybridoma cell whichproduces the mouse antibody of interest. The synthesized cDNA is clonedinto a vector such as a phage or a plasmid to obtain a cDNA library.Each of a recombinant phage or recombinant plasmid comprising a cDNAencoding VH and a recombinant phage or recombinant plasmid comprising acDNA encoding VL is isolated from the library by using a C region partor a V region part of an existing mouse antibody as the probe. Fullnucleotide sequences of VH and VL of the mouse antibody of interest onthe recombinant phage or recombinant plasmid are determined, and fulllength amino acid sequences of VH and VL are deduced from the nucleotidesequences.

As the non-human animal, any animal such as mouse, rat, hamster orrabbit can be used so long as a hybridoma cell can be preparedtherefrom.

The method for preparing total RNA from a hybridoma cell includes theguanidine thiocyanate-cesium trifluoroacetate method [Methods inEnzymology, 154, 3 (1987)] and the like, and the method for preparingmRNA from total RNA includes an oligo(dT)-immobilized cellulose columnmethod [Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Lab.,Press New York (1989)] and the like. In addition, a kit for preparingmRNA from a hybridoma cell includes Fast Track mRNA Isolation Kit(manufactured by Invitrogen), Quick Prep mRNA Purification Kit(manufactured by Pharmacia) and the like.

The method for synthesizing a cDNA and preparing a cDNA library includesthe usual methods [Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Lab., Press New York (1989), Current Protocols in MolecularBiology, Supplement 1-34], methods using a commercially available kitsuch as SuperScript™, Plasmid System for cDNA Synthesis and PlasmidCloning (manufactured by GIBCO BRL) or ZAP-cDNA Synthesis Kit(manufactured by Stratagene), and the like.

In preparing the cDNA library, the vector into which a cDNA synthesizedby using mRNA extracted from a hybridoma cell as the template isinserted may be any vector so long as the cDNA can be inserted. Examplesinclude ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK(+)[Nucleic Acids Research, 17, 9494 (1989)], λZAPII (manufactured byStratagene), λgt10 and λgt11 [DNA Cloning, A Practical Approach, I, 49(1985)], Lambda BlueMid (manufactured by Clontech), λExCell, pT7T3 18U(manufactured by Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)],pUC18 [Gene, 33, 103 (1985)] and the like.

As Escherichia coli into which the cDNA library constructed from a phageor plasmid vector is introduced, any Escherichia coli can be used, solong as the cDNA library can be introduced, expressed and maintained.Examples include XL1-Blue MRF′ [Strategies, 5, 81 (1992)], C600[Genetics, 39, 440 (1954)], Y1088 and Y1090 [Science, 222, 778 (1983)],NM522 [J. Mol. Biol., 166, 1 (1983)], K802 [J. Mol. Biol., 16, 118(1966)], JM105 [Gene, 38, 275 (1985)] and the like.

As the method for selecting a cDNA clone encoding VH and VL of anon-human animal antibody from the cDNA library, a colony hybridizationor a plaque hybridization using an isotope- or fluorescence-labeledprobe can be used [Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Lab., Press New York (1989)]. The cDNA encoding VH and VL canalso be prepared by preparing primers and carrying out polymerase chainreaction (hereinafter referred to as “PCR”; Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Lab., Press New York (1989);Current Protocols in Molecular Biology, Supplement 1-34) using a cDNAsynthesized from mRNA or a cDNA library as the template.

The nucleotide sequences of the cDNAs can be determined by digesting theselected cDNAs with appropriate restriction enzymes, cloning thefragments into a plasmid such as pBluescript SK(−) (manufactured byStratagene), carrying out the reaction of a generally used nucleotidesequence analyzing method such as the dideoxy method of Sanger et al.[Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] or the like and thenanalyzing the clones using an automatic nucleotide sequence analyzersuch as A.L.F. DNA Sequencer (manufactured by Pharmacia) or the like.

Whether or not the obtained cDNAs encode the full length amino acidsequences of VH and VL of the antibody containing a secretory signalsequence can be confirmed by deducing the full length amino acidsequences of VH and VL from the determined nucleotide sequence andcomparing them with the full length amino acid sequences of VH and VL ofknown antibodies [Sequences of Proteins of Immunological Interest, USDep. Health and Human Services (1991)].

(3) Analysis of Amino Acid Sequence of V Region of Non-Human AnimalAntibody

Regarding the full length amino acid sequences of VH and VL of theantibody comprising a secretory signal sequence, the length of thesecretory signal sequence and the N-terminal amino acid sequences can bededuced and subgroups to which they belong can also be found, bycomparing them with the full length amino acid sequences of VH and VL ofknown antibodies [Sequences of Proteins of Immunological Interest, USDep. Health and Human Services (1991)]. In addition, the amino acidsequences of each CDR of VH and VL can also be found by comparing themwith the amino acid sequences of VH and VL of known antibodies[Sequences of Proteins of Immunological Interest, US Dep. Health andHuman Services (1991)].

(4) Construction of Human Chimeric Antibody Expression Vector

A human chimeric antibody expression vector can be constructed bycloning cDNAs encoding VH and VL of a non-human animal antibody intoupstream of genes encoding CH and CL of a human antibody in the vectorfor expression of humanized antibody described in the item 2(1). Forexample, a human chimeric antibody expression vector can be constructedby ligating each of cDNAs encoding VH and VL of a non-human animalantibody to a synthetic DNA comprising nucleotide sequences at the3′-terminals of VH and VL of a non-human animal antibody and nucleotidesequences at the 5′-terminals of CH and CL of a human antibody and alsohaving a recognition sequence of an appropriate restriction enzyme atboth terminals, and by cloning them into upstream of genes encoding CHand CL of a human antibody contained in the vector for expression ofhumanized antibody described in the item 2(1) in such a manner that theycan be expressed in a suitable form.

(5) Construction of cDNA Encoding V Region of Human CDR-Grafted Antibody

cDNAs encoding VH and VL of a human CDR-grafted antibody can beconstructed as follows. First, amino acid sequences of the frameworks(hereinafter referred to as “FR”) of VH and VL of a human antibody forgrafting CDR of VH and VL of a non-human animal antibody of interest isselected. As the amino acid sequences of FRs of VH and VL of a humanantibody, any amino acid sequences can be used so long as they arederived from a human antibody. Examples include amino acid sequences ofFRs of VH and VL of human antibodies registered at databases such asProtein Data Bank, amino acid sequences common in each subgroup of FRsof VH and VL of human antibodies [Sequences of Proteins of ImmunologicalInterest, US Dep. Health and Human Services (1991)] and the like. Inorder to produce a human CDR-grafted antibody having enough activities,it is preferred to select an amino acid sequence having a homology ashigh as possible (at least 60% or more) with amino acid sequences of VHand VL of a non-human animal antibody of interest.

Next, the amino acid sequences of CDRs of VH and VL of the non-humananimal antibody of interest are grafted to the selected amino acidsequences of FRs of VH and VL of a human antibody to design amino acidsequences of VH and VL of the human CDR-grafted antibody. The designedamino acid sequences are converted into DNA sequences by considering thefrequency of codon usage found in nucleotide sequences of antibody genes[Sequences of Proteins of Immunological Interest, US Dep. Health andHuman Services (1991)], and the DNA sequences encoding the amino acidsequences of VH and VL of the human CDR-grafted antibody are designed.Based on the designed DNA sequences, several synthetic DNAs having alength of about 100 bases are synthesized, and PCR is carried out byusing them. In this case, it is preferred in each of the H chain and theL chain that 6 synthetic DNAs are designed in view of the reactionefficiency of PCR and the lengths of DNAs which can be synthesized.

Also, they can be easily cloned into the vector for expression ofhumanized antibody described in the item 2(1) by introducing recognitionsequences of an appropriate restriction enzyme into the 5′-terminals ofthe synthetic DNA present on both terminals. After the PCR, theamplified product is cloned into a plasmid such as pBluescript SK(−)(manufactured by Stratagene) and the nucleotide sequences are determinedby the method in the item 2(2) to thereby obtain a plasmid having DNAsequences encoding the amino acid sequences of VH and VL of the desiredhuman CDR-grafted antibody.

(6) Modification of Amino Acid Sequence of V Region of Human CDR-GraftedAntibody

It is known that when a human CDR-grafted antibody is prepared by simplygrafting only CDRs in VH and VL of a non-human animal antibody into FRsin VH and VL of a human antibody, its antigen-binding activity is lowerthan that of the original non-human animal antibody [BIO/TECHNOLOGY, 9,266 (1991)]. As the reason, it is considered that several amino acidresidues of FRs other than CDRs directly or indirectly relate toantigen-binding activity in VH and VL of the original non-human animalantibody, and that they are changed to different amino acid residues ofFRs in VH and VL of a human antibody. In order to solve the problem, inhuman CDR-grafted antibodies, among the amino acid sequences of FRs inVH and VL of a human antibody, an amino acid residue which directlyrelates to binding to an antigen, or an amino acid residue whichindirectly relates to binding to an antigen by interacting with an aminoacid residue in CDR or by maintaining the three-dimensional structure ofan antibody is identified and modified to an amino acid residue which isfound in the original non-human animal antibody to thereby increase theantigen binding activity which has been decreased [BIO/TECHNOLOGY, 9,266 (1991)].

In the preparation of a human CDR-grafted antibody, it is the mostimportant to efficiently identify the amino acid residues relating tothe antigen binding activity in FR. For identifying the amino acidresidues of FR relating to the antibody-antigen binding activity, thethree-dimensional structure of an antibody is constructed, and analyzedby X-ray crystallography [J. Mol. Biol., 112, 535 (1977)],computer-modeling [Protein Engineering, 7, 1501 (1994)] or the like.Although the information of the three-dimensional structure ofantibodies has been useful in the production of a human CDR-graftedantibody, method for producing a human CDR-grafted antibody which can beapplied to all antibodies has not been established yet. Therefore,various attempts must be currently be necessary, for example, severalmodified antibodies of each antibody are produced and the relationshipbetween each of the modified antibodies and its antibody bindingactivity is examined.

The amino acid sequence of FRs in VH and VL of a human antibody can bemodified using various synthetic DNA for modification according to PCRas described in the item 2(5). With regard to the amplified productobtained by the PCR, the nucleotide sequence is determined according tothe method as described in the item 2(2) to thereby confirm whether theobjective modification has been carried out.

(7) Construction of Human CDR-Grafted Antibody Expression Vector

A human CDR-grafted antibody expression vector can be constructed bycloning the cDNAs encoding VH and VL of the human CDR-grafted antibodyconstructed in the items 2(5) and (6) into upstream of the gene encodingCH and CL of a human antibody in the vector for expression of humanizedantibody described in the item 2(1). For example, a human CDR-graftedantibody expression vector can be constructed to be cloned byintroducing recognizing sequences of an appropriate restriction enzymeinto the 5′-terminal of synthetic DNAs positioned at both terminals,among the synthetic DNAs which are used in the items 2(5) and (6) forconstructing the VH and VL of the human CDR-grafted antibody, so thatthey are expressed in an appropriate form in upstream of the genesencoding CH and CL of a human antibody in the vector for expression ofhumanized antibody described in the item 2(1).

(8) Stable Production of Humanized Antibody

A transformant capable of stably producing a human chimeric antibody anda human CDR-grafted antibody (both hereinafter referred to as “humanizedantibody”) can be obtained by introducing the vector for humanizedantibody expression described in the items 2(4) and (7) into anappropriate animal cell.

The method for introducing a humanized antibody expression vector intoan animal cell includes electroporation [Japanese Published UnexaminedPatent Application No. 257891/90, Cytotechnology, 3, 133 (1990)] and thelike.

As the animal cell into which a humanized antibody expression vector isintroduced, any cell can be used so long as it is the cell of thepresent invention produced in the above item 1 and an animal cell whichcan produce the humanized antibody.

Examples include mouse myeloma cells such as NS0 cell and SP2/0 cell,Chinese hamster ovary cells such as CHO/dhfr⁻ cell and CHO/DG44 cell,rat myeloma such as YB2/0 cell and IR983F cell, BHK cell derived from asyrian hamster kidney, a human myeloma cell such as Namalwa cell, andthe like. Chinese hamster ovary cell CHO/DG44 cell and rat myeloma YB2/0cell are preferred.

After introduction of the humanized antibody expression vector, atransformant capable of stably producing the humanized antibody can beselected using a medium for animal cell culture comprising an agent suchas G418 sulfate (hereinafter referred to as “G418”; manufactured bySIGMA) and the like in accordance with the method disclosed in JapanesePublished Unexamined Patent Application No. 257891/90. The medium toculture animal cells includes RPMI 1640 medium (manufactured by NissuiPharmaceutical), GIT medium (manufactured by Nihon Pharmaceutical),EX-CELL 302 medium (manufactured by JRH), IMDM medium (manufactured byGIBCO BRL), Hybridoma-SFM medium (manufactured by GIBCO BRL), mediaobtained by adding various additives such as fetal bovine serum(hereinafter referred to as “FBS”) to these media, and the like. Thehumanized antibody can be produced and accumulated in the culturesupernatant by culturing the obtained transformant in a medium. Theamount of production and antigen binding activity of the humanizedantibody in the culture supernatant can be measured by a method such asenzyme-linked immunosorbent assay [hereinafter referred to as “ELISA”;Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter14 (1988); Monoclonal Antibodies: Principles and Practice, AcademicPress Limited (1996)] or the like. Also, the amount of the humanizedantibody produced by the transformant can be increased by using a DHFRgene amplification system in accordance with the method disclosed inJapanese Published Unexamined Patent Application No. 257891/90.

The humanized antibody can be purified from a culture supernatant of thetransformant using a protein A column [Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Chapter 8 (1988); Monoclonal Antibodies:Principles and Practice, Academic Press Limited (1996)]. In addition,purification methods generally used for the purification of proteins canalso be used. For example, the purification can be carried out throughthe combination of gel filtration, ion exchange chromatography,ultrafiltration, and the like. The molecular weight of the H chain, Lchain or antibody molecule as a whole of the purified humanized antibodycan be measured, e.g., by polyacrylamide gel electrophoresis[hereinafter referred to as “SDS-PAGE”; Nature, 227, 680 (1970)],Western blotting [Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, Chapter 12 (1988); Monoclonal Antibodies: Principles andPractice, Academic Press Limited (1996)] or the like.

B. Preparation of Fc Fusion Protein

(1) Construction of Fc Fusion Protein Expression Vector

An Fc fusion protein expression vector is an expression vector foranimal cell into which genes encoding the Fc region of a human antibodyand a protein to be fused are inserted, which can be constructed bycloning each of genes encoding the Fc region of a human antibody and theprotein to be fused into an expression vector for animal cell.

The Fc region of a human antibody includes those containing a part of ahinge region and/or CH1 in addition to regions containing CH2 and CH3regions. Also, it can be any Fc region so long as at least one aminoacid of CH2 or CH3 may be deleted, substituted, added or inserted, andsubstantially has the binding activity to the Fcγ receptor.

As the genes encoding the Fc region of a human antibody and the proteinto be fused, a chromosomal DNA comprising an exon and an intron can beused, and a cDNA can also be used. The method for linking the genes andthe Fc region includes PCR using each of the gene sequences as thetemplate (Molecular Cloning, Second Edition; Current Protocols inMolecular Biology, Supplement 1-34).

As the expression vector for animal cell, any vector can be used, solong as a gene encoding the C region of a human antibody can be insertedthereinto and expressed therein. Examples include pAGE107[Cytotechnology, 3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307(1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci.USA, 78, 1527 (1981), pSG1 β d2-4 [Cytotechnology, 4, 173 (1990)] andthe like. The promoter and enhancer in the expression vector for animalcell include SV40 early promoter and enhancer [J. Biochem., 101, 1307(1987)], Moloney mouse leukemia virus LTR promoter [Biochem. Biophys.Res. Commun., 149, 960 (1987)], immunoglobulin H chain promoter [Cell,41, 479 (1985)] and enhancer [Cell, 33, 717 (1983)], and the like.

(2) Preparation of DNA Encoding Fc Region of Human Antibody and Proteinto be Fused

A DNA encoding the Fc region of a human antibody and the protein to befused can be obtained in the following manner.

A cDNA is synthesized from mRNA extracted from a cell or tissue whichexpresses the protein of interest to be fused with Fc. The synthesizedcDNA is cloned into a vector such as a phage or a plasmid to obtain acDNA library. A recombinant phage or recombinant plasmid comprising acDNA encoding the protein of interest is isolated from the library byusing the gene sequence part of the protein of interest as the probe. Afull nucleotide sequence of the protein of interest on the recombinantphage or recombinant plasmid is determined, and a full length amino acidsequence is deduced from the nucleotide sequence.

As the non-human animal, any animal such as mouse, rat, hamster orrabbit can be used, so long as a cell or tissue can be removedtherefrom.

The method for preparing a total RNA from a cell or tissue includes theguanidine thiocyanate-cesium trifluoroacetate method [Methods inEnzymology, 154, 3 (1987)] and the like, and the method for preparingmRNA from total RNA includes an oligo (dT)-immobilized cellulose columnmethod (Molecular Cloning, Second Edition) and the like. In addition, akit for preparing mRNA from a cell or tissue includes Fast Track mRNAIsolation Kit (manufactured by Invitrogen), Quick Prep mRNA PurificationKit (manufactured by Pharmacia) and the like.

The method for synthesizing a cDNA and preparing a cDNA library includesthe usual methods (Molecular Cloning, Second Edition; Current Protocolsin Molecular Biology, Supplement 1-34); methods using a commerciallyavailable kit such as SuperScript™, Plasmid System for cDNA Synthesisand Plasmid Cloning (manufactured by GIBCO BRL) or ZAP-cDNA SynthesisKit (manufactured by Stratagene); and the like.

In preparing the cDNA library, the vector into which a cDNA synthesizedby using mRNA extracted from a cell or tissue as the template isinserted may be any vector so long as the cDNA can be inserted. Examplesinclude ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK(+)[Nucleic Acids Research, 17, 9494 (1989)], λZAPII (manufactured byStratagene), λgt10 and λgt11 [DNA Cloning, A Practical Approach, I, 49(1985)], Lambda BlueMid (manufactured by Clontech), λExCell, pT7T3 18U(manufactured by Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)],pUC18 [Gene, 33, 103 (1985)] and the like.

As Escherichia coli into which the cDNA library constructed from a phageor plasmid vector is introduced, any Escherichia coli can be used, solong as the cDNA library can be introduced, expressed and maintained.Examples include XL1-Blue MRF′ [Strategies, 5, 81 (1992)], C600[Genetics, 39, 440 (1954)], Y1088 and Y1090 [Science, 222, 778 (1983)],NM522 [J. Mol. Biol., 166, 1 (1983)], K802 [J. Mol. Biol., 16, 118(1966)], JM105 [Gene, 38, 275 (1985)] and the like.

As the method for selecting a cDNA clone encoding the protein ofinterest from the cDNA library, a colony hybridization or a plaquehybridization using an isotope- or fluorescence-labeled probe can beused (Molecular Cloning, Second Edition). The cDNA encoding the proteinof interest can also be prepared by preparing primers and using a cDNAsynthesized from mRNA or a cDNA library as the template according toPCR.

The method for fusing the protein of interest with the Fc region of ahuman antibody includes PCR. For example, any synthesized oligo DNAs(primers) are designed at the 5′-terminal and 3′-terminal of the genesequence encoding the protein of interest, and PCR is carried out toprepare a PCR product. In the same manner, any primers are designed forthe gene sequence encoding the Fc region of a human antibody to be fusedto prepare a PCR product. At this time, the primers are designed in sucha manner that the same restriction enzyme site or the same gene sequenceis present between the 3′-terminal of the PCR product of the protein tobe fused and the 5′-terminal of the PCR product of the Fc region. Whenit is necessary to modify the amino acids around the linked site,mutation is introduced by using the primer into which the mutation isintroduced. PCR is further carried out by using the two kinds of theobtained PCR fragments to link the genes. Also, they can be linked bycarrying out ligation after treatment with the same restriction enzyme.

The nucleotide sequence of the DNA can be determined by digesting thegene sequence linked by the above method with appropriate restrictionenzymes, cloning the fragments into a plasmid such as pBluescript SK(−)(manufactured by Stratagene), carrying out analysis by using a generallyused nucleotide sequence analyzing method such as the dideoxy method ofSanger et al. [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] or anautomatic nucleotide sequence analyzer such as A.L.F. DNA Sequencer(manufactured by Pharmacia).

Whether or not the obtained cDNA encodes the full length amino acidsequences of the Fc fusion protein containing a secretory signalsequence can be confirmed by deducing the full length amino acidsequence of the Fc fusion protein from the determined nucleotidesequence and comparing it with the amino acid sequence of interest.

(3) Stable Production of Fc Fusion Protein

A transformant capable of stably producing an Fc fusion protein can beobtained by introducing the Fc fusion protein expression vectordescribed in the item 2.B.(1) into an appropriate animal cell.

The method for introducing the Fc fusion protein expression vector intoan animal cell include electroporation [Japanese Published UnexaminedPatent Application No. 257891/90, Cytotechnology, 3, 133 (1990)] and thelike.

As the animal cell into which the Fc fusion protein expression vector isintroduced, any cell can be used, so long as it is the cell of thepresent invention prepared in the above item 1 and an animal cell whichcan produce the Fc fusion protein.

Examples include mouse myeloma cells such as NS0 cell and SP2/0 cell,Chinese hamster ovary cells such as CHO/dhfr⁻ cell and CHO/DG44 cell,rat myeloma such as YB2/0 cell and IR983F cell, BHK cell derived from asyrian hamster kidney, a human myeloma cell such as Namalwa cell, andthe like, and a Chinese hamster ovary cell CHO/DG44 cell, a rat myelomaYB2/0 cell and the like are preferred.

After introduction of the Fc fusion protein expression vector, atransformant capable of stably producing the Fc fusion proteinexpression vector can be selected using a medium for animal cell culturecomprising an agent such as G418 and the like in accordance with themethod disclosed in Japanese Published Unexamined Patent Application No.257891/90. The medium to culture animal cells includes RPMI 1640 medium(manufactured by Nissui Pharmaceutical), GIT medium (manufactured byNihon Pharmaceutical), EX-CELL 302 medium (manufactured by JRH), IMDMmedium (manufactured by GIBCO BRL), Hybridoma-SFM medium (manufacturedby GIBCO BRL), media obtained by adding various additives such as fetalbovine serum to these media, and the like. The Fc fusion protein can beproduced and accumulated in the culture supernatant by culturing theobtained transformant in a medium. The amount of production and antigenbinding activity of the Fc fusion protein in the culture supernatant canbe measured by a method such as ELISA. Also, the amount of the Fc fusionprotein produced by the transformant can be increased by using a dhfrgene amplification system in accordance with the method disclosed inJapanese Published Unexamined Patent Application No. 257891/90.

The Fc fusion protein can be purified from a culture supernatantculturing the transformant by using a protein A column or a protein Gcolumn (Antibodies, Chapter 8; Monoclonal Antibodies). In addition,purification methods generally used for the purification of proteins canalso be used. For example, the purification can be carried out throughthe combination of gel filtration, ion exchange chromatography,ultrafiltration and the like. The molecular weight as a whole of thepurified Fc fusion protein molecule can be measured by SDS-PAGE [Nature,227, 680 (1970)], Western blotting (Antibodies, Chapter 12, MonoclonalAntibodies) or the like.

Thus, methods for producing an antibody composition using an animal cellas the host cell have been described, but, as described above, theantibody composition can also be produced by yeast, an insect cell, aplant cell, an animal individual or a plant individual as describedabove.

When a host cell has a gene capable of expressing glycoprotein such asantibody molecule in the host cell, the host cell is prepared accordingto the method described in the item 1, the cell is cultured, and theglycoprotein of interest is purified from the culture to obtain theglycoprotein.

3. Activity Evaluation of Glycoprotein

Methods for measuring the amount of the purified glycoprotein, affinityto its receptor, half-life in blood, distribution in tissue afteradministration into blood and change of interactions between theproteins necessary for expression of pharmacological activity aremeasured by known methods described in Current Protocols In ProteinScience, John Wiley & Sons Inc., (1995); New Biochemical ExperimentationSeries 19—Animal Experimental Test, Tokyo Kagaku Dojin, edited byJapanese Biochemical Society (1991); New Biochemical ExperimentationSeries 8—Intracellular Information and Cell Response, Tokyo KagakuDojin, edited by Japanese Biochemical Society (1990); New BiochemicalExperimentation Series 9—Hormone I, Peptide hormone, Tokyo Kagaku Dojin,edited by Japanese Biochemical Society (1991); Experimental BiologicalCourse 3—Isotope Experimental Test, Maruzen (1982); MonoclonalAntibodies: Principles and Applications, Wiley-Liss, Inc., (1995);Enzyme-Linked Immuno Adsorbent Assay, 3rd Ed., Igaku Shoin (1987);Revised Enzyme Immunoassay, Gakusai Kikaku (1985); and the like.

Specific examples include a method in which a purified glycoprotein islabeled with a compound such as a radioisotope and binding activity to areceptor of the labeled glycoprotein or an interacted protein isquantitatively measured. Furthermore, interaction between the proteinscan be measured by using various apparatus such as BIAcore Seriesmanufactured by Biacore [J. Immunol. Methods, 145, 229 (1991);Experimental Medicine Supplement, Biomanual UP Series, Experimental Testof Intermolecular Interaction Experimental Test, Yodo-sha (1996)].

By administration of the labeled glycoprotein into the living body, thehalf-life in blood and the distribution in tissue after administeredinto the living body can be observed. Detection of the labeled body ispreferably carried out by a detection method in which a method fordetecting a labeled substance is combined with an antigen-antibodyreaction using an antibody specific to the glycoprotein which is to bedetected.

4. Activity Evaluation of Antibody Composition

As methods for measuring the amount of the protein, the affinity to anantigen and the effector function of the purified antibody composition,the known methods described in Monoclonal Antibodies, AntibodyEngineering and the like can be used.

As the examples, when the antibody composition is a humanized antibody,the binding activity with an antigen and the binding activity with anantigen-positive cultured cell clone can be measured by ELISA, theimmunofluorescent method [Cancer Immunol. Immunother., 36, 373 (1993)]or the like. The cytotoxic activity against an antigen-positive culturedcell clone can be evaluated by measuring CDC activity, ADCC activity[Cancer Immunol. Immunother., 36, 373 (1993)] and the like.

Also, safety and therapeutic effect of the antibody composition in humancan be evaluated using an appropriate model of animal species relativelyclose to human, such as Macaca fascicularis.

5. Analysis of Sugar Chains in Glycoprotein

The sugar chain structure of the glycoprotein expressed in a host cellcan be analyzed in accordance with the general analysis of the sugarchain structure of a glycoprotein. For example, the sugar chain which isbound to the IgG molecule comprises a neutral sugar such as galactose,mannose or fucose, an amino sugar such as N-acetylglucosamine and anacidic sugar such as sialic acid, and can be analyzed according to amethod such as sugar chain structure analysis by using sugar compositionanalysis, two dimensional sugar chain mapping method or the like.

Hereinafter, the analysis methods of the sugar chain in the antibodycomposition are specifically described, but other glycoproteins can beanalyzed in the same manner.

(1) Composition Analysis of Neutral Sugar and Amino Sugar

The composition of the sugar chain in an antibody molecule can beanalyzed by carrying out acid hydrolysis of sugar chains with an acidsuch as trifluoroacetic acid to release a neutral sugar or an aminosugar and measuring the composition ratio.

Examples include a method using a sugar composition analyzer (BioLC)manufactured by Dionex. The BioLC is an apparatus which analyzes a sugarcomposition by HPAEC-PAD (high performance anion-exchangechromatography-pulsed amperometric detection) method [J. Liq.Chromatogr., 6, 1577 (1983)].

The composition ratio can also be analyzed by a fluorescence labelingmethod using 2-aminopyridine. Specifically, the composition ratio can becalculated in accordance with a known method [Agric. Biol. Chem., 55(1),283-284 (1991)], by labeling an acid-hydrolyzed sample with afluorescence with 2-aminopyridylation and then analyzing the compositionby HPLC.

(2) Analysis of Sugar Chain Structure

The sugar chain structure in an antibody molecule can be analyzed by thetwo dimensional sugar chain mapping method [Anal. Biochem., 171, 73(1988), Biochemical Experimentation Methods 23—Methods for StudyingGlycoprotein Sugar Chains (Japan Scientific Societies Press) edited byReiko Takahashi (1989)]. The two dimensional sugar chain mapping methodis a method for deducing a sugar chain structure by, e.g., plotting theretention time or elution position of a sugar chain by reverse phasechromatography as the X-axis and the retention time or elution positionof the sugar chain by normal phase chromatography as the Y-axis,respectively, and comparing them with those of known sugar chains.

Specifically, sugar chains are released from an antibody by subjectingthe antibody to hydrazinolysis, and the released sugar chains aresubjected to fluorescence labeling with 2-aminopyridine (hereinafterreferred to as “PA”) [J. Biochem., 95, 197 (1984)], and then the sugarchains are separated from an excess PA-treating reagent by gelfiltration, and subjected to reverse phase chromatography. Thereafter,each peak of the separated sugar chains is subjected to normal phasechromatography. The sugar chain structure can be deduced by plotting theresults on a two dimensional sugar chain map and comparing them with thespots of a sugar chain standard (manufactured by Takara Shuzo) or aliterature [Anal. Biochem., 171, 73 (1988)].

The structure deduced by the two dimensional sugar chain mapping methodcan be confirmed by further carrying out mass spectrometry such asMALDI-TOF-MS of each sugar chain.

6. Analysis of Side Effect of Introduced RNA

The introduced RNA might affect the level of expression, translation andthe like of a gene having high homology, in addition to the inhibitionof the function of a target enzyme [Nature Biotechnol., 21, 635 (2003),Proc. Natl. Acad. Sci. USA, 101, 1892 (2004)]. Accordingly, when aglycoprotein such as an antibody composition is produced, whether or notgrowth of a transformant or expression of a produced glycoprotein isinfluenced by side effect of the introduction of RNA should be analyzed.

Specifically, regarding the expression cell of a glycoprotein such as anantibody composition, a parent cell into which the RNA of the presentinvention is not introduced and the introduced cell are culturedsimultaneously to confirm that there is no change in the growth curve ofthe cells and the level of expression of the glycoprotein. By analyzingthe various properties of the produced glycoproteins, it is confirmedthat the produced glycoprotein have no difference except for thebiological activity due to the difference in the sugar chain structures.

The sugar chain structure of the produced glycoprotein can be analyzedby the method described in the above item 5. Various properties of theglycoprotein can be analyzed by any known analysis methods of proteins.The analysis methods of proteins include physicochemical analyses suchas electrophoresis, gel filtration, isoelectric point and amino acidsequence analyses, affinity to an antigen in the case where the producedglycoprotein is an antibody, enzyme activity in the case where theproduced glycoprotein is an enzyme, and affinity to a respective ligandor receptor in the case where the glycoprotein is a ligand or areceptor.

7. Application of Glycoprotein

A glycoprotein such as an antibody composition has a sugar chainstructure with which no fucose is modified and has high biologicalactivity so that effects such as improvement of affinity to itsreceptor, improvement of half-life in blood, improvement of distributionin tissue after administration in blood and improvement of interactionbetween a protein necessary for expression of pharmacological activityare expected. Particularly, the antibody composition has high effectorfunction, i.e., high antibody-dependent cell-mediated cytotoxicactivity. The glycoprotein having high physiological activity or theantibody composition having high ADCC activity is useful for preventingand treating various diseases including cancers, inflammatory diseases,immune diseases such as autoimmune diseases and allergies,cardiovascular diseases and viral or bacterial infections.

In the case of cancers, namely malignant tumors, cancer cells grow.General anti-tumor agents inhibit the growth of cancer cells. Incontrast, an antibody having high ADCC activity can treat cancers byinjuring cancer cells through its cell killing effect, and therefore, itis more effective as a therapeutic agent than the general anti-tumoragents. At present, in the therapeutic agent for cancers, an anti-tumoreffect of an antibody medicament alone is insufficient in many cases, sothat combination therapy with chemotherapy has been carried out[Science, 280, 1197 (1998)]. If higher anti-tumor effect is found by theantibody composition of the present invention alone, the dependency onchemotherapy will be decreased and side effects will be reduced.

In immune diseases such as inflammatory diseases, autoimmune diseasesand allergies, in vivo reactions of the diseases are induced by therelease of a mediator molecule by immunocytes, so that the allergicreaction can be inhibited by eliminating immunocytes using an antibodyhaving high ADCC activity.

The cardiovascular diseases include arteriosclerosis and the like. Thearteriosclerosis is treated using balloon catheter at present, butcardiovascular diseases can be prevented and treated by suppressinggrowth of arterial cells in restricture after treatment using anantibody having high ADCC activity.

Various diseases including viral and bacterial infections can beprevented and treated by suppressing proliferation of cells infectedwith a virus or bacterium using an antibody having high ADCC activity.

An antibody which recognizes a tumor-related antigen, an antibody whichrecognizes an allergy- or inflammation-related antigen, an antibodywhich recognizes cardiovascular disease-related antigen, an antibodywhich recognizes an autoimmune disease-related antigen or an antibodywhich recognizes a viral or bacterial infection-related antigen areexemplified below.

The antibody which recognizes a tumor-related antigen includesanti-CA125 antibody, anti-17-1A antibody, anti-integrin αvβ3 antibody,anti-CD33 antibody, anti-CD22 antibody, anti-HLA antibody, anti-HLA-DRantibody, anti-CD20 antibody, anti-CD19 antibody, anti-EGF receptorantibody [Immunology Today, 21, 403 (2000)], anti-CD10 antibody[American Journal of Clinical Pathology, 113, 374 (2000); Proc. Natl.Acad. Sci. USA, 79, 4386 (1982)], anti-GD₂ antibody [Anticancer Res.,13, 331 (1993)], anti-GD₃ antibody [Cancer Immunol. Immunother., 36, 260(1993)], anti-GM₂ antibody [Cancer Res., 54, 1511 (1994)], anti-HER2antibody [Proc. Natl. Acad. Sci. USA, 89, 4285 (1992)], anti-CD52antibody [Nature, 332, 323 (1988)], anti-MAGE antibody [British J.Cancer, 83, 493 (2000)], anti-HM1.24 antibody [Molecular Immunol., 36,387 (1999)], anti-parathyroid hormone-related protein (PTHrP) antibody[Cancer, 88, 2909 (2000)], anti-FGF8 antibody [Proc. Natl. Acad. Sci.USA, 86, 9911 (1989)], anti-basic fibroblast growth factor antibody,anti-FGF8 receptor antibody [J. Biol. Chem., 265, 16455 (1990)],anti-basic fibroblast growth factor receptor antibody, anti-insulin-likegrowth factor antibody, anti-insulin-like growth factor receptorantibody [J. Neurosci. Res., 40, 647 (1995)], anti-PMSA antibody [J.Urology, 160, 2396 (1998)], anti-vascular endothelial cell growth factorantibody [Cancer Res., 57, 4593 (1997)], anti-vascular endothelial cellgrowth factor receptor antibody [Oncogene, 19, 2138 (2000)] and thelike.

The antibody which recognizes an allergy- or inflammation-relatedantigen includes anti-IgE antibody, anti-CD23 antibody, anti-CD11aantibody [Immunology Today, 21, 403 (2000)], anti-CRTH2 antibody [J.Immunol., 162, 1278 (1999)], anti-CCR8 antibody (WO99/25734), anti-CCR3antibody (U.S. Pat. No. 6,207,155), anti-interleukin 6 antibody[Immunol. Rev., 127, 5 (1992)], anti-interleukin 6 receptor antibody[Molecular Immunol., 31, 371 (1994)], anti-interleukin 5 antibody[Immunol. Rev., 127, 5 (1992)], anti-interleukin 5 receptor antibody,anti-interleukin 4 antibody [Cytokine, 3, 562 (1991)], anti-interleukin4 receptor antibody [J. Immunol. Meth., 217, 41 (1998)], anti-tumornecrosis factor antibody [Hybridoma, 13, 183 (1994)], anti-tumornecrosis factor receptor antibody [Molecular Pharmacol., 58, 237(2000)], anti-CCR4 antibody [Nature, 400, 776 (1999)], anti-chemokineantibody [J. Immuno. Meth., 174, 249 (1994)], anti-chemokine receptorantibody [J. Exp. Med., 186, 1373 (1997)] and the like.

The antibody which recognizes a cardiovascular disease-related antigenincludes anti-GpIIb/IIIa antibody [J. Immunol., 152, 2968 (1994)],anti-platelet-derived growth factor antibody [Science, 253, 1129(1991)], anti-platelet-derived growth factor receptor antibody [J. Biol.Chem., 272, 17400 (1997)], anti-blood coagulation factor antibody[Circulation, 101, 1158 (2000)] and the like.

The antibody which recognizes an antigen relating to autoimmune diseasessuch as psoriasis, rheumatoid arthritis, crohn' disease, uncreativecolitis, systemic lupus erythema tosus, and multiple sclerosis includesan anti-auto-DNA antibody [Immunol. Letters, 72, 61 (2000)], anti-CD11aantibody, anti-ICAM3 antibody, anti-CD80 antibody, anti-CD2 antibody,anti-CD3 antibody, anti-CD4 antibody, anti-integrin α4β7 antibody,anti-CD40L antibody, anti-IL-2 receptor antibody [Immunology Today, 21,403 (2000)] and the like.

The antibody which recognizes a viral or bacterial infection-relatedantigen includes anti-gp120 antibody [Structure, 8, 385 (2000)],anti-CD4 antibody [J. Rheumatology, 25, 2065 (1998)], anti-CCR5 antibodyand anti-Vero toxin antibody [J. Clin. Microbiol., 37, 396 (1999)] andthe like.

These antibodies can be obtained from public organizations such as ATCC(The American Type Culture Collection), RIKEN Gene Bank at The Instituteof Physical and Chemical Research and National Institute of Bioscienceand Human Technology, Agency of Industrial Science and Technology, orprivate reagent sales companies such as Dainippon Pharmaceutical, R & DSYSTEMS, PharMingen, Cosmo Bio and Funakoshi.

Hereinafter, examples of the Fc region fusion protein of the presentinvention are described below.

Examples of a fusion protein of an binding protein relating toinflammatory diseases and immune diseases such as autoimmune diseasesand allergies with the Fc region of an antibody include etanercept whichis a fusion protein of sTNFRII with the Fc region (U.S. Pat. No.5,605,690), alefacept which is a fusion protein of LFA-3 expressed onantigen presenting cells with the Fc region (U.S. Pat. No. 5,914,111), afusion protein of Cytotoxic T Lymphocyte-associated antigen-4 (CTLA-4)with the Fc region [J. Exp. Med., 181, 1869 (1995)], a fusion protein ofinterleukin 15 with the Fc region [J. Immunol., 160, 5742 (1998)], afusion protein of factor VII with the Fc region [Proc. Natl. Acad. Sci.USA, 98, 12180 (2001)], a fusion protein of interleukin 10 with the Fcregion [J. Immunol., 154, 5590 (1995)], a fusion protein of interleukin2 with the Fc region [J. Immunol., 146, 915 (1991)], a fusion protein ofCD40 with the Fc region [Surgery, 132, 149 (2002)], a fusion protein ofFlt-3 (fms-like tyrosine kinase) with the antibody Fc region [Acta.Haemato., 95, 218 (1996)], a fusion protein of OX40 with the antibody Fcregion [J. Leu. Biol., 72, 522 (2002)] and the like. In addition, manyfusion proteins have been reported, such as various human CD molecules[CD2, CD30 (TNFRSF8), CD95 (Fas), CD106 (VCAM-1), CD137], adhesionmolecules [ALCAM (activated leukocyte cell adhesion molecule),cadherins, ICAM (intercellular adhesion molecule)-1, ICAM-2, ICAM-3],cytokine receptors (hereinafter “receptor” being referred to as “R”),(interleukin-4R, interleukin-5R, interleukin-6R, interleukin-9R,interleukin-10R, interleukin-12R, interleukin-13Rα1, interleukin-13Rα2,interleukin-15R, interleukin-21R), chemokines, cell death-inducingsignal molecules [B7-H1, DR6 (Death receptor 6), PD-1 (Programmeddeath-1), TRAIL R1], costimulating molecules [B7-1, B7-2, B7-H2, ICOS(inducible co-stimulator)], growth factors (ErbB2, ErbB3, ErbB4, HGFR),diffentiation-inducing factors (B7-H3), activating factors (NKG2D),signal transfer molecules (gp130), or receptors or ligands of thesebinding proteins with the antibody Fc region.

The medicament comprising the glycoprotein such as the antibodycomposition can be administered as a therapeutic agent alone, butgenerally, it is preferred to provide it as a pharmaceutical formulationproduced by an appropriate method well known in the technical field ofpharmaceutics, by mixing it with at least one pharmaceuticallyacceptable carrier.

It is preferred to select a route of administration which is mosteffective in treatment. Examples include oral administration andparenteral administration, such as buccal, tracheal, rectal,subcutaneous, intramuscular or intravenous administration. In the caseof a glycoprotein preparation, intravenous administration is preferred.

The dosage form includes sprays, capsules, tablets, granules, syrups,emulsions, suppositories, injections, ointments, tapes and the like.

The pharmaceutical preparation suitable for oral administration includesemulsions, syrups, capsules, tablets, powders, granules and the like.

Liquid preparations such as emulsions and syrups can be produced using,as additives, water; sugars such as sucrose, sorbitol and fructose;glycols such as polyethylene glycol and propylene glycol; oils such assesame oil, olive oil and soybean oil; antiseptics such asp-hydroxybenzoic acid esters; flavors such as strawberry flavor andpeppermint; and the like.

Capsules, tablets, powders, granules and the like can be produced using,as additives, fillers such as lactose, glucose, sucrose and mannitol;disintegrating agents such as starch and sodium alginate; lubricantssuch as magnesium stearate and talc; binders such as polyvinyl alcohol,hydroxypropylcellulose and gelatin; surfactants such as fatty acidester; plasticizers such as glycerin; and the like.

The pharmaceutical preparation suitable for parenteral administrationincludes injections, suppositories, sprays and the like.

Injections can be prepared using a carrier such as a salt solution, aglucose solution or a mixture of both thereof. Also, powdered injectionscan be prepared by freeze-drying the glycoprotein in the usual way andadding sodium chloride thereto.

Suppositories can be prepared using a carrier such as cacao butter,hydrogenated fat or carboxylic acid.

Sprays can be prepared using the glycoprotein as such or using ittogether with a carrier which does not stimulate the buccal or airwaymucous membrane of the patient and can facilitate absorption of theglycoprotein by dispersing it as fine particles.

The carrier includes lactose, glycerol and the like. Depending on theproperties of the glycoprotein and the carrier, it is possible toproduce pharmaceutical preparations such as aerosols and dry powders. Inaddition, the components exemplified as additives for oral preparationscan also be added to the parenteral preparations.

Although the dose or the frequency of administration varies depending onthe objective therapeutic effect, administration method, treatingperiod, age, body weight and the like, it is usually 10 μg/kg to 20mg/kg per day and per adult.

Also, as the method for examining antitumor effect of the antibodycomposition against various tumor cells, in vitro tests include CDCactivity measuring method, ADCC activity measuring method and the like,and in vivo tests include antitumor experiments using a tumor system inan experimental animal such as a mouse.

CDC activity and ADCC activity and antitumor experiments can be carriedout in accordance with the methods described in literature [CancerImmunology Immunotherapy, 36, 373 (1993); Cancer Research, 54, 1511(1994)] and the like.

The present invention is described below in detail based on Examples;however, Examples are only simple illustrations, and the scope of thepresent invention is not limited thereto.

EXAMPLE 1

Preparation of Lectin-Resistant CHO/DG44 Cell by IntroducingGMD-Targeting Small Interfering RNA (siRNA) Expression Plasmid:

1. Construction of GMD-Targeting siRNA Expression Vector

(1) Cloning of “Human U6 Promoter-Cloning Site-Terminator” SequenceExpression Cassette

A “human U6 promoter-cloning site-terminator” sequence expressioncassette was obtained according to the following procedure (FIG. 1).

First, a forward primer in which recognition sequences of restrictionenzymes HindIII and EcoRV were added to the 5′-terminal of a nucleotidesequence which binds to human U6 promoter sequence [GenBank Acc. No.M14486] (hereinafter referred to as “hU6p-F-HindIII/EcoRV”, representedby SEQ ID NO:59) and a reverse primer in which recognition sequences ofrestriction enzymes XbaI and EcoRV, continued 6 adenines basescorresponding to a terminator sequence, and recognition sequences ofrestriction enzymes KpnI and SacI for insertion of a different syntheticoligonucleotide DNA to the 5′-terminal of a nucleotide sequence whichbinds to human U6 promoter sequence (hereinafter referred to as“hU6p-R-term-XbaI/EcoRV”, represented by SEQ ID NO:60) were designed.

Then, after preparing 50 μL of a reaction solution [KOD buffer #1(manufactured by TOYOBO), 0.1 mmol/L dNTPs, 1 mmol/L MgCl₂, 0.4 μmol/Lprimer hU6p-F-HindIII/EcoRV, and 0.4 μmol/L primerhU6p-R-term-XbaI/EcoRV] containing 40 ng of U6_FUT8_B_puro plasmid,described in Example 12 of WO03/085118, as a template, polymerase chainreaction (hereinafter referred to as “PCR”) was carried out using DNApolymerase KOD polymerase (manufactured by TOYOBO). After heating at 94°C. for 2 minutes, PCR was carried out by 30 cycles, one cycle consistingof reaction at 94° C. for 15 seconds, reaction at 65° C. for 5 secondsand reaction at 74° C. for 30 seconds.

After the PCR, the reaction solution was subjected to agarose gelelectrophoresis, and a specifically amplified fragment (about 300 bp)was recovered using RECOCHIP (manufactured by TAKARA BIO). The DNAfragment was dissolved in 30 μL of NEBuffer 2 (manufactured by NewEngland Biolabs), and digested for 2 hours at 37° C. with 10 units ofrestriction enzymes XbaI (manufactured by New England Biolabs) andHindIII (manufactured by New England Biolabs). The reaction solution waspurified by phenol/chloroform extraction and ethanol precipitation, andthe recovered DNA fragments digested with the restriction enzymes weredissolved in 20 μL of sterilized water.

Also, 1 μg of plasmid pBluescript II KS(+) (manufactured by STRATAGENE)was dissolved in 30 μL of NEBuffer 2 (manufactured by New EnglandBiolabs) containing 100 μg/mL BSA (manufactured by New England Biolabs),and digested for 8 hours at 37° C. with 10 units of restriction enzymesHindIII and XbaI (manufactured by New England Biolabs). After thedigestion reaction, 22 μL of sterilized water, 6 μL of 10× alkalinephosphatase buffer, and 1 unit of alkaline phosphatase E. coli C75(manufactured by TAKARA BIO) were added to the reaction solution forcarrying out dephosphorylation reaction at 37° C. for 1 hour. Thereaction solution was subjected to agarose gel electrophoresis, andHindIII-XbaI fragment (about 2.9 kb) derived from plasmid pBluescript IIKS(+) was recovered using RECOCHIP (manufactured by TAKARA BIO).

Then, 8 μL of the DNA fragment (about 300 bp) and 2 μL of HindIII-XbaIfragment (about 2.9 kb) derived from plasmid pBluescript II KS(+)obtained above were mixed with 10 μL of Ligation High (manufactured byTOYOBO), and were allowed to react for 2 hours at 16° C. E. coli DH5α(manufactured by TOYOBO) was transformed with the reaction solution, anda plasmid was isolated from the resulting ampicillin-resistant clonesusing QIAprep spin Mini prep Kit (manufactured by Qiagen). The plasmidis hereinafter referred to as “pBS-U6term”.

(2) Ligation of “Human U6 Promoter-Cloning Site-Terminator” SequenceExpression Cassette to pPUR

The “human U6 promoter-cloning site-terminator” sequence expressioncassette in the plasmid pBS-U6term obtained in the above (1) wasexcised, and ligated to expression vector pPUR (manufactured byCLONTECH) according to the following procedure (FIG. 2).

First, 1 μg of plasmid pBS-U6term prepared in the above (1) above wasdissolved in 20 μL of NEBuffer 2 (manufactured by New England Biolabs)containing 100 μg/mL BSA (manufactured by New England Biolabs), anddigested with 10 units of a restriction enzyme EcoRV (manufactured byNew England Biolabs) for 2 hours at 37° C. The reaction solution wassubjected to agarose gel electrophoresis, and a DNA fragment containing“human U6 promoter-cloning site-terminator” sequence expression cassette(about 350 bp) was recovered using RECOCHIP (manufactured by TAKARABIO).

Also, 6 μg of plasmid pPUR (manufactured by CLONTECH) was dissolved in20 μL of NEBuffer 2 (manufactured by New England Biolabs), and digestedwith 10 units of a restriction enzyme PvuII (manufactured by New EnglandBiolabs) for 2 hours at 37° C. After the reaction, 5 μL of sterilizedwater, 3 μL of 10× alkaline phosphatase buffer, and 1 unit of alkalinephosphatase E. coli C75 (manufactured by TAKARA BIO) were added to thereaction solution for carrying out dephosphorylation reaction at 37° C.for 1 hour. The reaction solution was subjected to agarose gelelectrophoresis, and a PvuII fragment (about 4.3 kb) derived fromplasmid pPUR was recovered using RECOCHIP (manufactured by TAKARA BIO).

Then, 8 μL of the DNA fragment (about 350 bp) containing human U6promoter-cloning site-terminator sequence expression cassette and 2 μLof the PvuII fragment (about 4.3 kb) derived from plasmid pPUR obtainedabove were mixed with 10 μL of Ligation High (manufactured by TOYOBO)and allowed to react for 3 hours at 16° C. E. coli DH5α (manufactured byTOYOBO) was transformed with the reaction solution, and plasmid DNAswere isolated from the resulting ampicillin-resistant clones usingQIAprep spin Mini prep Kit (manufactured by Qiagen). Approximately 0.5μg of the plasmid DNA was dissolved in 10 μL of NEBuffer 2 (manufacturedby New England Biolabs), and digested with 10 units of restrictionenzymes SacI and HindIII (manufactured by New England Biolabs) for 2hours at 37° C. The reaction solution was subjected to agarose gelelectrophoresis to confirm the presence and inserted direction of theinserted fragment of interest. In addition, nucleotide sequences of theDNA inserted into each plasmid were determined using DNA sequencer 377(manufactured by Perkin Elmer) and BigDye Terminator v3.0 CycleSequencing Kit (manufactured by Applied Biosystems) according to themanufacturer's instruction. pPUR PvuII-seq-F (SEQ ID NO:61) and pPURPvuII-seq-R (SEQ ID NO:62) were used as primers for sequence analysis toconfirm that the inserted DNA fragments had the identical human U6promoter sequence to GenBank Acc. No. M14486 and that primer sitesequences used to amplify the “human U6 promoter-cloningsite-terminator” sequence expression cassette and ligation sitesequences were correct, and a plasmid in which the inserted hU6promoters was in the same direction as the puromycin-resistant geneexpression unit selected from the resulting plasmids. The plasmid ishereinafter referred to as “pPUR-U6term”.

(3) Selection of Target Sequence and Design of Synthetic Oligo DNA

Synthetic oligo DNAs which form a double-stranded DNA cassettecontaining siRNA target sequence against the GMD gene of Chinese hamsterovary CHO/DG44 cell were prepared as follows.

First, seven target sequences which satisfied the conditions describedbelow were selected from Chinese hamster-derived GMD cDNA sequence(GenBank Acc. No. AF525364; SEQ ID NO: 8). Selected target sequences arerepresented by SEQ ID NOs: 36 to 42.

-   Condition 1: A consensus sequence consisting of “NAR(N17)YNN” is    included, wherein N represents A, G, U or C; R represents A or G,    and Y represents U or C, respectively.-   Condition 2: Condition 1 is satisfied, and a sequence of continued 3    or more same bases is not included.-   Condition 3: Conditions 1 and 2 are satisfied, and GC content is    more preferably 35 to 45%, and preferably 45 to 55%.-   Condition 4: A sequence of 29 bases in which 6 bases are added to    the 5′- or 3′-terminal of 23 bases which satisfy the conditions 1-3    satisfies the condition 2.-   Condition 5: The condition 4 is satisfied, and GC content is more    preferably 35 to 45%, and preferably 45 to 55%.

In addition, double-stranded DNA cassettes were designed for theselected target sequences according to the following procedure.Sequentially from 5′-terminal, the double-stranded DNA cassettes have3′-cohesive end generated by digestion with a restriction enzyme SacI,sense DNA that corresponds to the SEQ ID NOs:36 to 42, loop sequence ofhuman miR-23-precursor-19 micro RNA consisting 10 bases (GenBank Acc.No. AF480558), an antisense DNA complementary to the DNA sequences ofSEQ ID NOs: 36 to 42, and 3′-cohesive end generated by a restrictionenzyme KpnI. The 5′-terminal of the double-stranded DNA wasphosphorylated.

The nucleotide sequence of the sense strand of the synthetic oligo DNA(hereinafter referred to as “GMD-dsRNA-A-F”) which was designed based onthe target sequence represented by SEQ ID NO:36 is represented by SEQ IDNO:43; the nucleotide sequence of the antisense strand (hereinafterreferred to as “GMD-dsRNA-A-R”) is represented by SEQ ID NO:44; thenucleotide sequence of the sense strand sequence of the synthetic oligoDNA (hereinafter referred to as “GMD-dsRNA-B-F”) which was designedbased on the target sequence represented by SEQ ID NO:37 is representedby SEQ ID NO:45; the nucleotide sequence of the antisense strand(hereinafter referred to as “GMD-dsRNA-B-R”) is represented by SEQ IDNO:46; the nucleotide sequence of the sense strand of the syntheticoligo DNA (hereinafter referred to as “GMD-dsRNA-C-F”) which wasdesigned based on the target sequence represented by SEQ ID NO:38 isrepresented by SEQ ID NO:47; the nucleotide sequence of the antisensestrand (hereinafter referred to as “GMD-dsRNA-C-R”) is represented bySEQ ID NO:48; the nucleotide sequence of the sense strand of thesynthetic oligo DNA (hereinafter referred to as “GMD-dsRNA-D-F”) whichwas designed based on the target sequence represented by SEQ ID NO:39 isrepresented by SEQ ID NO:49; the nucleotide sequence of the antisensestrand (hereinafter referred to as “GMD-dsRNA-D-R”) is represented bySEQ ID NO:50; the nucleotide sequence of the sense strand of thesynthetic oligo DNA (hereinafter referred to as “GMD-dsRNA-E-F”) whichwas designed based on the target sequence represented by SEQ ID NO:40 isrepresented by SEQ ID NO:51; the nucleotide sequence of the antisensestrand (hereinafter referred to as “GMD-dsRNA-E-R”) is represented bySEQ ID NO:52; the nucleotide sequence of the sense strand of thesynthetic oligo DNA (hereinafter referred to as “GMD-dsRNA-F-F”) whichwas designed based on the target sequence represented by SEQ ID NO:41 isrepresented by SEQ ID NO:53; the nucleotide sequence of the antisensestrand (hereinafter referred to as “GMD-dsRNA-F-R”) in SEQ ID NO:54; thenucleotide sequence of the sense strand of the synthetic oligo DNA(hereinafter referred to as “GMD-dsRNA-F-F”) which was designed based onthe target sequence represented by SEQ ID NO:42 is represented by SEQ IDNO:55; and the nucleotide sequence of the antisense strand (hereinafterreferred to as “GMD-dsRNA-F-R”) is represented by SEQ ID NO:56,respectively. Designed synthetic oligo DNAs were synthesized accordingto the conventional procedure (Molecular Cloning, Second Edition).

(4) Insertion of Synthetic Oligo DNA into Plasmid pPUR-U6Term

The synthetic oligo DNAs synthesized in the above (3) were inserted intothe cloning site of pPUR-U6term obtained in the above (2) (FIG. 3).

First, synthetic oligo DNAs were annealed according to the followingprocedure. In 10 μL of an annealing buffer [10 mmol/L Tris (pH 7.5)-50mmol/L NaCl-1 mmol/L EDTA], 200 pmol each of sense and antisense strandsof the synthetic oligo DNAs were dissolved, followed by boiling for 2minutes. Then, they were cooled gradually to room temperature overapproximately 3 hours. Subsequently, annealed synthetic oligo DNAs werediluted 15-fold with sterilized water.

Also, 3 μg of plasmid pPUR-U6term was dissolved in 40 μL of NEBuffer 1(manufactured by New England Biolabs) containing 100 μg/mL BSA(manufactured by New England Biolabs), and digested with 20 units ofrestriction enzymes KpnI and SacI (manufactured by New England Biolabs)for 4 hours at 37° C. After the digestion reaction, 12 μL of sterilizedwater, 6 μL of 10× alkaline phosphatase buffer, and 1 unit of alkalinephosphatase E. coli C75 (manufactured by TAKARA BIO) were added to thereaction solution for carrying out dephosphorylation reaction at 37° C.for 1 hour. The reaction solution was subjected to agarose gelelectrophoresis, and a KpnI-SacI fragment (about 4.5 kb) derived fromplasmid pPUR-U6term was recovered using RECOCHIP (manufactured by TAKARABIO).

Then, 1 μL of the double-stranded synthetic oligo solution and 1 μL ofthe KpnI-SacI fragment derived from plasmid pPUR-U6term obtained abovewere mixed with 8 μL of sterilized water and 10 μL of Ligation High(manufactured by TOYOBO), and allowed to react overnight at 16° C. E.coli DH5α (manufactured by TOYOBO) was transformed with the reactionsolution, and plasmid DNAs were isolated from the resultingampicillin-resistant clones using QIAprep spin Mini prep Kit(manufactured by Qiagen).

The nucleotide sequences of the DNA inserted into each plasmid weredetermined using DNA sequencer 377 (manufactured by Perkin Elmer) andBigDye Terminator v3.0 Cycle Sequencing Kit (manufactured by AppliedBiosystems) according to the manufacturer's instruction. A plasmid DNAwhich was boiled for approximately 1 minute and cooled rapidly was usedas a template, and pPUR PvuII-seq-F (SEQ ID NO:61), hU6p Tsp45I/seq-F(SEQ ID NO:63) and pPUR PvuII-seq-R (SEQ ID NO:62) were used as primersfor sequence analysis, and the inserted synthetic oligo DNA sequencesand ligation sites were confirmed. Hereinafter, a plasmid into which adouble-stranded DNA consisting of synthetic oligo DNA GMD-dsRNA-A-F andGMD-dsRNA-A-R are introduced is referred to as “pPUR/GMDshA”; a plasmidinto which a double-stranded DNA consisting of synthetic oligo DNAGMD-dsRNA-B-F and GMD-dsRNA-B-R are introduced is referred to as“pPUR/GMDshB”; a plasmid into which double-stranded DNA consisting ofsynthetic oligo DNA GMD-dsRNA-C-F and GMD-dsRNA-C-R are introduced isreferred to as “pPUR/GMDshC”; a plasmid into which a double-stranded DNAconsisting of synthetic oligo DNA GMD-dsRNA-D-F and GMD-dsRNA-D-R areintroduced is referred to as “pPUR/GMDshD”; a plasmid into which adouble-stranded DNA consisting of synthetic oligo DNA GMD-dsRNA-E-F andGMD-dsRNA-E-R are introduced is referred to as “pPUR/GMDshE”; a plasmidinto which a double-stranded DNA consisting of synthetic oligo DNAGMD-dsRNA-F-F and GMD-dsRNA-F-R are introduced is referred to as“pPUR/GMDshF”; and a plasmid into which a double-stranded DNA consistingof synthetic oligo DNA GMD-dsRNA-G-F and GMD-dsRNA-G-R are introduced isreferred to as “pPUR/GMDshG”.

2. Obtaining and Culture of Lectin-Resistant Clones by IntroducingGMD-Targeting siRNA Expression Vector

(1) Obtaining of Lectin-Resistant Clones by Introducing GMD-TargetingsiRNA Expression Vector

Each of plasmids pPUR/GMDshA, pPUR/GMDshB, pPUR/GMDshC, pPUR/GMDshD,pPUR/GMDshE, pPUR/GMDshF and pPUR/GMDshG constructed in the item 1 ofthis Example was introduced into CHO/DG44 cell-derived anti-CCR4chimeric antibody-producing clone, clone 35-02-12 (hereinafter referredto as “clone 32-05-12”), which was obtained by the same method asdescribed in Reference Example 1 of WO03/85118, and clones resistant toa lectin which recognizes a sugar chain structure in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in a N-glycoside-linked sugar chain, a Lens culinarisagglutinin (hereinafter referred to as “LCA”) were obtained as describedbelow.

Transfection of various siRNA expression vector plasmids into clone32-05-12 was carried out by electroporation [Cytotechnology, 3, 133(1990)] according to the following procedure. First, 10 μg of each ofsiRNA expression vector plasmids was dissolved in 30 μL of NEBuffer 4(manufactured by New England Biolabs), and digested to be linearizedwith 10 units of a restriction enzyme FspI (manufactured by New EnglandBiolabs) overnight at 37° C. After the linearized plasmid was confirmedby agarose gel electrophoresis using a part of the reaction solution,the remaining reaction solution was purified by phenol/chloroformextraction and ethanol precipitation, and the recovered linearizedplasmid was dissolved in 10 μL of sterilized water.

Also, clone 32-05-12 was suspended in a K-PBS buffer (137 mmol/L KCl,2.7 mmol/L NaCl, 8.1 mmol/L Na₂HPO₄, 1.5 mmol/KH₂PO₄, and 4.0 mmol/LMgCl₂) at 8×10⁶ cells/mL. After 200 μL of the cell suspension (1.6×10⁶)was mixed with 10 μL of the above linearized plasmid solution, all ofthe cell/DNA mixture was transferred to Gene Pulser Cuvette (Electrodeinterval: 2 mm) (manufactured by BIO-RAD), and transfection was carriedout under conditions of 350V pulse voltage and 250 μF capacitance usinga cell fusion device Gene Pulser (manufactured by BIO-RAD). After thetransfection, the cell suspension was suspended in a basal medium[Iscove's Modified Dulbecco's Medium (hereinafter referred to as “IMDM”,manufactured by Invitrogen) containing 10% fetal bovine dialyzed serum(manufactured by Invitrogen), 50 μg/mL gentamicin (manufactured byNacalai Tesque), and 500 nmol/L MTX (manufactured by SIGMA)], andinoculated into four 10 cm-dishes for adherent cell culture(manufactured by Falcon). After culturing under conditions of 5% CO₂ and37° C. for 24 hours, the culture supernatant was removed, and a basalmedium supplemented with 12 μg/mL puromycin (manufactured by SIGMA) wasadded thereto, followed by culturing for further 7 days. Subsequently,the culture supernatant was removed from one of the dishes, a basalmedium containing 12 μg/mL puromycin (manufactured by SIGMA) was addedthereto, followed by culturing for further 6 to 8 days, and appearedpuromycin-resistant colonies were counted. Also, the culture supernatantwas removed from the remaining dishes, and a basal medium supplementedwith 12 μg/mL puromycin (manufactured by SIGMA) and 0.5 mg/mL LCA(manufactured by VECTOR) was added thereto, followed by culturing forfurther 7 days. As a result, lectin-resistant clones were obtained whenpPUR/GMDshB was introduced.

(2) Expansion Culture of Lectin-Resistant Clones

Lectin-resistant clones obtained in the above (1) by introducingpPUR/GMDshB were expansion cultured according to the followingprocedure.

First, the number of appeared colonies in each dish was counted. Then,lectin-resistant colonies were scraped and sucked up with a pipetteman(manufactured by GILSON) under observation with a stereoscopicmicroscope, and collected onto a U-shaped-bottom 96-well plate foradherent cells (manufactured by ASAHI TECHNOGLASS). After trypsintreatment, each clone was dispensed onto a flat-bottom 96-well plate foradherent cells (manufactured by Greiner), and cultured in a basal mediumcontaining 12 μg/mL puromycin (manufactured by SIGMA) under conditionsof 5% CO₂ and 37° C. for a week. After the culture, 10 clones wereexpansion cultured in a basal medium containing 12 μg/mL puromycin(manufactured by SIGMA). Clones used in the expansion culture wererespectively named “12-GMDB-1”, “12-GMDB-2”, “12-GMDB-3”, “12-GMDB-4”,“12-GMDB-5”, “12-GMDB-6”, “12-GMDB-7”, “12-GMDB-8”, “12-GMDB-9” and“12-GMDB-10”, and used in the analysis described in the item 3 below.Also, clone 12-GMDB-5 has been deposited to International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology (Tsukuba Central 6, 1, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan) as FERM BP-10051 on Jul. 1, 2004.

3. Determination of the Amount of GMD mRNA in Lectin-Resistant Cloneinto which GMD-Targeting siRNA Expression Vector was Introduced

(1) Preparation of Total RNA

A total RNA was prepared from clone 32-05-12 and lectin-resistant cloneswhich were obtained in the item 2 of this Example according to thefollowing procedure. Clone 32-05-12 was suspended in a basal medium andlectin-resistant clones were suspended in a basal medium supplementedwith 12 μg/mL puromycin (manufactured by SIGMA) at a density of 3×10⁵cells/mL, and they inoculated at 4 ml into 6 cm-dishes for adherentcells (manufactured by Falcon). Cells were statically cultured underconditions of 5% CO₂ and 37° C. for 3 days, and each cell suspension wascollected after trypsin treatment, and centrifuged at 1,000 rpm and 4°C. for 5 minutes to remove the supernatant. After the cells weresuspended in Dulbecco's PBS buffer (manufactured by Invitrogen) andcentrifuged again at 1,000 rpm and 4° C. for 5 minutes to remove thesupernatant, a total RNA was extracted using RNeasy (manufactured byQIAGEN). The method was carried out according to the manufacturer'sinstruction, and the prepared total RNA was dissolved in 40 μL ofsterilized water.

(2) Synthesis of Single-Stranded cDNA

A single-stranded cDNA was synthesized from 3 μg of each of the totalRNA obtained in the item (1) by reverse transcription reaction witholigo (dT) primer in 20 μL reaction system using SUPERSCRIPT™Preamplification System for First Strand cDNA Synthesis (manufactured byInvitrogen) according to the manufacturer's instruction. Subsequently,the reaction solution was treated with RNase and the final reactionvolume was adjusted to 40 μL. In addition, each of the reactionsolutions was diluted 50-fold with sterilized water, and used fordetermination of the amount of gene transcription described below.

(3) Determination of the Amount of Gene Transcription by SYBR-PCR

The amount of mRNA transcribed from GMD gene and β-actin gene weredetermined using For Real Time PCR TaKaRa Ex Taq R-PCR Version(manufactured by TAKARA BIO) according to the following procedure.

In this connection, plasmid pAGE249GMD containing CHO cell-derived GMDcDNA described in Example 15 of WO02/31140, each diluted atconcentration of 0.0512 fg/μL, 0.256 fg/μL, 1.28 fg/μL, 6.4 fg/μL, 32fg/μL and 160 fg/μL were used as internal controls of GMD determination;β-actin standard plasmid described in Example 9 of WO02/31140, eachdiluted at concentration of 1.28 fg/μL, 6.4 fg/μL, 32 fg/μL, 160 fg/μL,800 fg/μL and 4,000 fg/μL were used as internal controls of β-actindetermination. As PCR primers, forward and reverse primers representedby SEQ ID NOs: 64 and 65, respectively, were used to amplify GMD, andforward and reverse primers represented by SEQ ID NOs: 66 and 67,respectively, were used to amplify β-actin gene.

Then, 20 μL of reaction solution [R-PCR buffer (manufactured by TAKARABIO), 2.5 mmol/L Mg²⁺ Solution for R-PCR (manufactured by TAKARA BIO),0.3 mmol/L dNTP mixture (manufactured by TAKARA BIO), 0.3 μmol/L forwardprimer, 0.3 μmol/L reverse primer, 2×10⁻⁵ diluted SYBR GreenI, 1 unitTaKaRa Ex Taq R-PCR] containing 5 μL of the single-stranded cDNAprepared in the item (2) or each concentration of internal controlplasmid solution was prepared using For Real Time PCR TaKaRa Ex TaqR-PCR Version (manufactured by TAKARA BIO). The prepared reactionsolutions were dispensed into each well of a 96-well Polypropylene PCRPlate (manufactured by Falcon), and the plate was sealed with PlateSealer (manufactured by Edge Biosystems). ABI PRISM 7700 SequenceDetection System was used for PCR and analysis, and the amount of GMDmRNA and the amount of β-actin mRNA were determined according to themanufacturer's instruction.

A calibration curve was made based upon the measurements with theinternal control plasmid, and the amount of GMD mRNA and the amount ofβ-actin mRNA were converted into numerical terms. In addition, assumingthat the amount of mRNA transcribed from β-actin gene are uniform amongthe clones, the relative amount of GMD mRNA to the amount of β-actinmRNA were calculated and compared, and the results are shown in FIG. 4.

It was shown that the amount of GMD mRNA in all the clones obtained byintroducing the GMD-targeting siRNA expression plasmid were reduced to8% to 50% in that of the parent cell.

EXAMPLE 2

Production of Antibody Composition Using Lectin-Resistant CHO/DG44 Cellinto which GMD-Targeting siRNA Expression Plasmid was Introduced:

1. Obtaining of Antibody Compositions Produced by Lectin-Resistant Cloneinto which GMD-Targeting siRNA Expression Plasmid was Introduced

Anti-CCR4 chimeric antibodies produced by lectin-resistant clone12-GMDB-2 and clone 12-GMDB-5 into which the GMD-targeting siRNAexpression plasmid was introduced obtained in Example 1 were obtainedaccording to the following procedure.

Clone 32-05-12 was suspended in a basal medium and clones 12-GMDB-2 and12-GMDB-5 were suspended in a basal medium supplemented with 12 μg/mLpuromycin (manufactured by SIGMA) at a density of 3×10⁵ cells/mL, andthey were inoculated at 15 mL into a T75 flask for adherent cells(manufactured by Greiner). After culturing under conditions of 5% CO₂and 37° C. for 6 days, the culture supernatant was removed, and afterwashing twice with 10 mL of Dulbecco's PBS (manufactured by Invitrogen),20 mL of EXCELL301 medium (manufactured by JRH Bioscience) was added.After culturing under conditions of 5% CO₂ and 37° C. for 7 days, theculture supernatant was recovered, and anti-CCR4 chimeric antibodieswere purified using a MabSelect column (manufactured by AmershamBioscience) according to the manufacturer's instruction. After exchangewith 10 mmol/L KH₂PO₄ buffer using Econo-Pac 10DG (manufactured by BioRad), anti-CCR4 chimeric antibodies purified from culture supernatant ofvarious clones were subjected to sterile filtration by using Millex GV(manufactured by MILLIPORE) of 0.22 mm pore size.

2. Composition Analysis of Monosaccharide of Antibody CompositionsProduced by Lectin-Resistant Clone into which GMD-Targeting siRNAExpression Plasmid was Introduced

Composition analysis of monosaccharide was carried out on the anti-CCR4chimeric antibodies obtained in the item 1 of this Example according tothe known method [Journal of Liquid Chromatography, 6, 1577 (1983)].TABLE 1 Ratio of sugar chains in Clone which fucose is not bound32-05-12  3% 12-GMDB-2 78% 12-GMDB-5 79%

The composition ratio of complex type sugar chains in which fucose isnot bound among the total complex sugar chains calculated from thecomposition of monosaccharide ratio of each antibody is shown inTable 1. Among antibody compositions produced from parent clone32-05-12, which was used to introduce the GMD-targeting siRNA expressionvector, the ratio of sugar chains in which fucose is not bound was 3%,while those of 12-GMDB-2 and 12-GMDB-5 lectin-resistant clones intowhich siRNA was introduced were 78% and 79%, respectively, demonstratingthat the ratios of sugar chains in which fucose is not bound are greatlyincreased in comparison with the parent cell.

The above results demonstrate that the α1,6-fucose content in theantibodies produced by the host cells can be controlled by theintroduction of GMD-targeting siRNA.

EXAMPLE 3

Serum-Free Fed-Batch Culture of Lectin-Resistant CHO/DG44 Cell intowhich GMD-Targeting siRNA Expression Plasmid was Introduced:

1. Adaptation of Lectin-Resistant CHO/DG44 Cell into which GMD-TargetingsiRNA Expression Plasmid was Introduced to Serum-Free Medium

The parent clone 32-05-12 before vector introduction, andlectin-resistant clones 12-GMDB-2 and 12-GMDB-5 into which theGMD-targeting siRNA expression plasmid were introduced obtained inExample 1 were adapted to a serum-free medium according to the followingprocedure.

Clone 32-05-12 was suspended in a basal medium and clones 12-GMDB-2 and12-GMDB-5 were suspended in a basal medium supplemented with 12 μg/mLpuromycin (manufactured by SIGMA) at a density of 3×10⁵ cells/mL, andeach was inoculated at 5 ml into a T75 flask for adherent cells(manufactured by Greiner). After culturing under conditions of 5% CO₂and 37° C. for 3 days, cell suspension was obtained by trypsintreatment, and cells were recovered by centrifugation at 1,000 rpm for 5minutes. Clone 32-05-12 was suspended in EX-CELL302 medium (manufacturedby JRH) containing 500 nmol/L MTX (manufactured by SIGMA), 6 mmol/LL-glutamine (manufactured by Invitrogen), 50 μg/mL gentamicin(manufactured by Nacalai Tesque) and 100 nmol/L3,3,5-triiodo-L-thyronine (manufactured by SIGMA) (hereinafter referredto as “serum-free medium”) and clones 12-GMDB-2 and 12-GMDB-5 weresuspended in the serum-free medium supplemented with 12 μg/mL puromycin(manufactured by SIGMA) at a density of 5×10⁵ cells/mL, and 15 mL of thecell suspension was inoculated into a 125 mL conical flask (manufacturedby Corning). After ventilating the flask with 5% CO₂ (at least 4-foldvolume of culture vessel) and sealing the flask, suspension rotationculture was carried out at 90-100 rpm and 35° C. Passage was repeated at3 to 4 day intervals, and finally, clones which could grow in theserum-free medium were obtained. Hereinafter, clones 32-05-12, 12-GMDB-2and 12-GMDB-5 adapted to the serum-free medium were referred to as“32-05-12AF”, and 12-GMDB-2 and 12-GMDB-5 adapted to the serum-freemedium were referred to as “12-GMDB-2AF” and “12-GMDB-5AF”,respectively.

2. Serum-Free Fed-Batch Culture of Lectin-Resistant CHO/DG44 Cell intowhich GMD-Targeting siRNA Expression Plasmid was Introduced and Adaptedto Serum-Free Medium

(1) Serum-Free Fed-Batch Culture in Conical Flask

Serum-free fed-batch culture was carried out using clones 32-05-12AF,12-GMDB-2AF, and 12-GMDB-5AF adapted to the serum-free medium in theitem 1 of this Example according to the following procedure.

EX-CELL302 medium (manufactured by JRH) containing 500 nmol/L MTX(manufactured by SIGMA), 6 mmol/L L-glutamine (manufactured byInvitrogen), 100 nmol/L 3,3,5-triiodo-L-thyronine (manufactured bySIGMA), 0.1% Pluronic F-68 (manufactured by Invitrogen), and 5,000 mg/LD(+)-glucose (manufactured by Nacalai Tesque) (hereinafter referred toas “serum-free fed-batch medium”) was used for fed-batch culture, and amedium containing amino acids prepared at higher concentrations thanusual addition (0.177 g/L L-alanine, 0.593 g/L L-argininemonohydrochloride, 0.177 g/L L-asparagine monohydrate, 0.212 g/LL-asparatic acid, 0.646 g/L L-cystine dihydrochloride, 0.530 g/LL-glutamic acid, 5.84 g/L L-glutamine, 0.212 g/L glycine, 0.297 g/LL-histidine monohydrochloride dihydrate, 0.742 g/L L-isoleucine, 0.742g/L L-leucine, 1.031 g/L L-lysine monohydrochloride, 0.212 g/LL-methionine, 0.466 g/L L-phenylalanine, 0.283 g/L L-proline, 0.297 g/LL-serine, 0.671 g/L L-threonine, 0.113 g/L L-tryptophan, 0.735 g/LL-tyrosine disodium dihydrate, and 0.664 g/L L-valine), vitamins (0.0918mg/L d-biotin, 0.0283 g/L D-calcium pantothenate, 0.0283 g/L cholinechloride, 0.0283 g/L folic acid, 0.0509 g/L myo-inositol, 0.0283 g/Lniacinamide, 0.0283 g/L pyridoxal hydrochloride, 0.00283 g/L riboflavin,0.0283 g/L thiamine hydrochloride, and 0.0918 mg/L cyanocobalamin) and0.314 g/L insulin (hereinafter referred to as “feed medium”) was used asa medium for feeding.

Clones 32-05-12AF, 12-GMDB-2AF and 12-GMDB-5AF were suspended in theserum-free fed-batch medium at a density of 3×10⁵ cells/mL, and 40 mLeach of the cell suspension was inoculated into a 250 mL conical flask(manufactured by Corning). After ventilating the flask with 5% CO₂ (atleast 4-fold volume of culture vessel), and sealing the flask,suspension rotation culture was carried out at 90 to 100 rpm and 35° C.On days 3, 6, 9 and 12 after starting the culture, 3.3 mL of feed mediumwas added to supplement the consumption of amino acids and the like, and20% (w/v) glucose solution was added at a final concentration of 5,000mg/L to adjust the glucose concentration. On days 0, 3, 6, 9, 12 and 14after starting the culture, 2-4 mL each of the culture was collected,and used for the analyses described in the items (2) to (4). Inaddition, the fed-batch culture was finished on day 14 after startingthe culture, and the whole culture was collected and used for theanalysis described in the item (4).

(2) Measurement of the Viable Cell Number

Viable cell number and viability of the culture in the item (1),collected on days 0, 3, 6, 9, 12 and 14 after starting the culture, weremeasured by trypan blue staining. Viable cell number at each point oftime after starting the culture of clones 32-05-12AF, 12-GMDB-2AF and12-GMDB-5AF are shown in FIG. 5. Clone 12-GMDB-2 grew slower incomparison with clone 32-05-12AF, and retained a high viability even onday 14. Also, the viable cell number of clone 12-GMDB-5AF at each pointof time after starting the culture was similar to those of clone32-05-12. Therefore, it was demonstrated that the target sequences ofGMD-targeting siRNA had no significant effects on the cell growth.

(3) Determination of Antibody Concentration

Antibody concentrations contained in the culture supernatants on days 0,3, 6, 9, 12 and 14 after starting the culture obtained in the item (1)were determined according to the following procedure.

In 750 mL of Dulbecco's PBS (manufactured by Invitrogen), 1 mL ofanti-human IgG (H+L) antibody (manufactured by American Qualex) wasdissolved, and the mixture was dispensed at 50 μl onto each well of anELISA plate. After leaving overnight at 4° C., the solution was removed,and 100 μL of PBS containing 1% BSA (bovine serum albumin) (hereinafterreferred to as “BSA-PBS”) was added to each well, and the plate wasallowed to stand for approximately 1 hour at room temperature, andstored at −20° C. On measuring the amount of antibody, the plate wasthawed at room temperature, and after removing the BSA-PBS in wells, 50μL of the culture supernatant diluted with BSA-PBS was added to eachwell. After the plate was allowed to stand for 1 to 2 hours at roomtemperature, the wells were washed with PBS containing 0.05% Tween20™(hereinafter referred to as “Tween-PBS”). After removing the washingliquid, 50 μL of goat anti-human IgG (H&L)-HRP (manufactured by AmericanQualex) diluted 2000-fold with BSA-PBS, was added to each well as asecond antibody. After the plate was allowed to stand for 1 to 2 hoursat room temperature, wells were washed with Tween-PBS and then withresin water. After washing, 50 μL of an ABTS substrate solutionsupplemented with 0.1% H₂O₂ [0.55 g of2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)ammonium wasdissolved in 1 L of 0.1 mol/L citrate buffer (pH 4.2), and supplementedwith oxygenated water at 1 μL/mL before use] was added to each well forcolor development. After the plate was allowed to stand at roomtemperature for approximately 15 minutes, when appropriate colordeveloped, 50 μL of 5% SDS solution was added to each well to stop thereaction. Absorption at 490 nm was measured with that of at 415 nm asreference using a microplate reader. Antibody concentrations of eachdiluted sample were calculated using the linear area of the sigmoidcurve of the calibration curve prepared with a standard of purifiedantibody preparation. Each antibody concentration of culturesupernatants was calculated by multiplying the antibody concentrationsof the obtained diluted samples by the dilution rate. Determinationresults of the antibody concentrations in the culture supernatants ateach point of time after starting culture of clones, 32-05-12AF,12-GMDB-2AF and 12-GMDB-5AF, are shown in FIG. 6. The antibodycomposition concentrations in the culture supernatants after start ofculture were similar among clones 32-05-12AF, 12-GMDB-2AF and12-GMDB-5AF. Therefore, it was demonstrated that the GMD-targeting siRNAsequence does not affect cellular antibody productivity.

(4) Sugar Chain Structural Analysis of Antibody Compositions

Anti-CCR4 chimeric antibody compositions were purified from culturesupernatants of the serum-free fed-batch culture on day 14 of clone32-05-12AF and those of the serum-free fed-batch culture on days 6, 12and 14 of clones 12-GMDB-2AF and 12-GMDB-5AF obtained in the item (1) byusing a MabSelect column (manufactured by Amersham Biosciences)according to the manufacturer's instruction. After exchange with 10mmol/L KH₂PO₄ buffer using Econo-Pac 10DG (manufactured by Bio Rad),anti-CCR4 chimeric antibody compositions purified from culturesupernatants of various clones were subjected to sterile filtration byusing Millex GV (manufactured by MILLIPORE) of 0.22 mm pore size.Composition analysis of monosaccharide was carried out with theanti-CCR4 chimeric antibody compositions obtained from culturesupernatants of each clones adapted to the serum-free medium accordingto the known method [Journal of Liquid Chromatography, 6, 1577 (1983)].The ratio of sugar chains in which fucose is not bound among the totalcomplex sugar chains (hereinafter referred to as “fucose(−)%”)calculated from composition ratio of monosaccharide of each antibodycompositions is shown in Table 2. TABLE 2 Clone Culturing days Fucose(−)% 32-05-12AF 14  7% 12-GMDB-2AF 6 88% 12 87% 14 85% 12-GMDB-5AF 6 84% 1282% 14 81%

On day 14 when the culture was finished, fucose(−)% of antibodycompositions produced by clone 32-05-12AF was 7%, while those oflectin-resistant clones 12-GMDB-2AF and 12-GMDB-5AF into which theGMD-targeting siRNA expression plasmid was introduced were 81 to 85%.Therefore, it was demonstrated that, also in the serum-free fed-batchmedium, lectin-resistant clones into which the GMD-targeting siRNAexpression plasmid was introduced could produce antibody compositionshaving higher fucose(−)% than the parent clone. In addition, fucose(−)%of antibody compositions produced by clones 12-GMDB-2AF and 12-GMDB-5AFshowed roughly constant values on days 6, 12, and 14. Therefore, it wasdemonstrated that inhibitory effects on α1,6-fucose addition to complexsugar chains of antibody compositions by introduction of theGMD-targeting siRNA expression plasmid were stable in the serum-freefed-batch culture.

EXAMPLE 4

Screening of siRNA Target Sequence Effective for ObtainingLectin-Resistant Clone Using α1,6-Fucosyltransferase (FUT8)-TargetingsiRNA Expression Vector Library and Construction of EffectiveFUT8-Targeting siRNA Expression Vector:

1. Construction of α1,6-Fucosyltransferase (FUT8)-Targeting siRNAExpression Vector Library (FUT8shRNAlib/pPUR)

(1) Obtaining of CHO Cell-Derived α1,6-Fucosyltransferase (FUT8) cDNASequence

A cDNA encoding α1,6-fucosyltransferase (FUT8) was cloned from asingle-stranded cDNA prepared from Chinese hamster ovary-derivedCHO/DG44 cell according to the procedure described in WO00/61739.

First, 5′-untranslated region-specific forward primer (SEQ ID NO: 68)and 3′-untranslated region-specific reverse primer (SEQ ID NO: 69) weredesigned based upon the nucleotide sequence of mouse FUT8 cDNA (GenBankAcc. No. AB025198).

Then, after preparing 25 μL of a reaction solution [ExTaq buffer(manufactured by TaKaRa), 0.2 mmol/L dNTPs, 4% DMSO, and 0.5 μmol/Lspecific primers described above (SEQ ID NOs: 68 and 69)] containing 1μL of CHO/DG44 cell-derived single-stranded cDNA, PCR was carried outusing DNA polymerase ExTaq (manufactured by TaKaRa). After heating at94° C. for 1 minute, the PCR was carried out by 30 cycles, one cycleconsisting of reaction at 94° C. for 30 seconds, reaction at 55° C. for30 seconds and reaction at 72° C. for 2 minutes, followed by reaction at72° C. for 10 minutes.

After the PCR, the reaction solution was subjected to 0.8% agarose gelelectrophoresis, and a specifically amplified fragment (about 2 kb) wasrecovered. The DNA fragment was ligated to plasmid pCR2.1 using TOPO TAcloning Kit (manufactured by Invitrogen) according to the manufacturer'sinstruction, and E. coli DH5α was transformed with the ligationsolution. Among the resulting kanamycin-resistant colonies, plasmid DNAswere isolated from 8 clones with which cDNA was inserted according tothe known method.

After reaction using BigDye Terminator Cycle Sequencing FS ReadyReaction Kit (manufactured by Applied Biosystems) according to themanufacturer's instruction, the sequence of cDNA inserted in eachplasmid were analyzed using DNA sequencer ABI PRISM 377 manufactured byApplied Biosystems. By this method, it was confirmed that all the cDNAsinserted in the plasmids were cDNA encoding full-length Chinese hamsterFUT8 ORF. Among cDNA inserted in the plasmid DNAs whose sequences weredetermined, a plasmid DNA, free of readout errors of nucleotideresulting from PCR, was selected. Hereinafter, the plasmid is referredto as “CHfFUT8-pCR2.1”. The nucleotide sequence of Chinese hamster FUT8cDNA, determined in this manner, is represented by SEQ ID NO: 1.

(2) Preparation of FUT8-Targeting siRNA Expression Vector Library

Human tRNA-val promoter type FUT8-targeting siRNA expression vectorlibrary was constructed using CHfFUT8-pCR2.1 obtained in the item (1),based upon the method described in Example 13 of WO03/46186. Also, pPUR(manufactured by CLONTECH) was used as a vector, using a recognitionsequence of a restriction enzyme BamHI as a loop sequence betweenantisense and sense DNAs. Hereinafter, the prepared library is referredto as “FUT8shRNAlib/pPUR/DH10B”.

Plasmid vectors were prepared by amplifying the siRNA expression vectorlibrary, FUT8shRNAlib/pPUR/DH10B. LB agar medium containing 100 μg/mLampicillin was prepared using sterilized dishes [243 mm×243 mm×18 mm(manufactured by Nalgenunc)], and 50 μL/dish FUT8shRNAlib/pPUR/DH10Bglycerol stock was plated. After stationary culture overnight at 37° C.,the E. coli on the plates were collected in suspension with sterilizedwater, and a plasmid DNA was recovered according to the known method.Hereinafter, the recovered plasmid is referred to as“FUT8shRNAlib/pPUR”.

2. Obtaining of Lectin-Resistant Clone into whichα1,6-Fucosyltransferase (FUT8)-Targeting siRNA Expression Library wasIntroduced

FUT8-targeting siRNA expression library plasmid, FUT8shRNAlib/pPURobtained in the item 1 of this Example was introduced into clone32-05-12, and clones resistant to LCA, a lectin which specificallyrecognizes α1,6-fucose, were isolated as follows.

Plasmid FUT8shRNAlib/pPUR obtained in the item 1 of this Example wasdigested with a restriction enzyme FspI (manufactured by New EnglandBiolabs) to be linearized, and after 10 μg of the linearized plasmidFUT8shRNAlib/pPUR was introduced into 1.6×10⁶ cells of clone 32-05-12 byelectroporation [Cytotechnology, 3, 133 (1990)], the cells weresuspended in a basal medium [IMDM (manufactured by Invitrogen)containing 10% fetal bovine serum (manufactured by Invitrogen), 50 μg/mLgentamicin (manufactured by Nacalai Tesque), and 500 nmol/L MTX(manufactured by SIGMA)], and inoculated at 8 mL into 3 dishes of 10 cmfor adherent cell culture (manufactured by Falcon). Also, transfectionwas carried out 10 times under the same conditions, and the cells werecultured in a total of 30 culture dishes of 10 cm. After culturing in a5% CO₂ incubator at 37° C. for 24 hours, the medium was exchanged with 8mL of a basal medium containing 12 μg/mL puromycin (manufactured bySIGMA). After culturing in a 5% CO₂ incubator at 37° C. for 7 days, themedium was exchanged with 8 mL of a basal medium containing 12 μg/mLpuromycin (manufactured by SIGMA) and 0.5 mg/mL LCA (manufactured byVECTOR), and the culture was continued for further 6 to 8 days toisolate lectin-resistant clones.

3. Analysis of Target Sequence of α1,6-Fucosyltransferase(FUT8)-Targeting siRNA Expression Plasmid

(1) Isolation of siRNA Expression Cassette on Genomic DNA ofLectin-Resistant Clone

siRNA expression cassette was isolated from genomic DNA oflectin-resistant clones obtained in the item 2 of this Example asfollows (FIG. 7).

Lectin-resistant clones were collected into a flat-bottom plate foradherent cells (manufactured by Greiner) according to the known method[Gene Targeting, Oxford University Press (1993)], and cultured in abasal medium containing 12 μg/mL puromycin (manufactured by SIGMA) at37° C. for 1 week in the 5% CO₂ incubator.

After culturing, each clone of the plate was treated with trypsin, anddispensed onto 2 flat-bottom 96-well plates for adherent cells(manufactured by Greiner). One plate was used as a replica plate, andanother was freeze-stored as a master plate. After the replica plate wascultured in a basal medium containing 12 μg/mL puromycin (manufacturedby SIGMA) at 37° C. for 1 week in a 5% CO₂ incubator, genomic DNA wasprepared from each clone according to the known method [AnalyticalBiochemistry, 201, 331 (1992)], and dissolved in 30 μL each of TE-RNasebuffer (pH 8.0) [10 mmol/L Tris-HCl, 1 mmol/L EDTA, and 200 μg/mL RNaseA] overnight, then diluted at 0.05 μg/μL with sterilized water.

In addition, a forward primer which binds to the upstream of thetRNA-val promoter region of the siRNA expression cassette (SEQ ID NO:70)and a reverse primer which binds to the downstream of the terminatorsequence of the siRNA expression cassette (SEQ ID NO:71) were eachdesigned for FUT8-targeting siRNA expression plasmid, FUT8shRNAlib/pPUR.

Polymerase chain reaction (PCR) was carried out with DNA polymerase KODpolymerase (manufactured by TOYOBO), using the genomic DNA prepared fromeach clone as a template. A reaction solution (50 μL) [KOD Buffer1(manufactured by TOYOBO), 0.2 mmol/L dNTPs, 1 mmol/L MgCl₂, and 0.4μmol/L of the above primers (SEQ ID NOs:70 and 71)] containing 5 μL ofthe genomic DNA solution described above was prepared for each clone,and after heating at 94° C. for 1 minute, the PCR was carried out by 25cycles, one cycle consisting of reaction at 97° C. for 10 seconds andreaction at 68° C. for 30 seconds. After the PCR, the reaction solutionwas subjected to agarose gel electrophoresis, and the amplified fragment(about 300 bp) containing the siRNA expression cassette region wasrecovered.

Also, 2 μg of plasmid pPUR (manufactured by CLONTECH) was digested witha restriction enzyme PvuII (manufactured by New England Biolabs) at 37°C. overnight. After the digestion reaction, the reaction solution wassubjected to agarose gel electrophoresis, and a digested DNA fragment(about 4.3 kb) was recovered.

The PCR-amplified fragment (about 300 bp) obtained above was ligated toa PvuII fragment derived from plasmid pPUR using Ligation High(manufactured by TOYOBO). E. coli DH5α was transformed with the reactionsolution. Plasmid DNAs were isolated from a number of obtainedampicillin-resistant colonies according to the known method.

(2) Analysis of Target Sequence Contained in the siRNA Expression Unit

FUT8-targeting sequences contained in the siRNA expression cassette ofthe plasmids obtained in the item (1) were analyzed

First, after reaction with BigDye Terminator v3.0 Cycle sequencing Kit(manufactured by Applied Biosystems) according to the manufacturer'sinstruction, nucleotide sequences of siRNA expression cassette whichwere inserted into each plasmid DNA obtained in the item (1) wereanalyzed using DNA sequencer ABI PRISM 377 (manufactured by AppliedBiosystems). Among nucleotide sequences determined for 159 clones,homology of target sequences against FUT8 was compared with the sequenceof CHO cell FUT8 cDNA (SEQ ID NO:1), and the start and end points ofeach target sequence, which corresponded to SEQ ID NO:1, are shown inTable 3. TABLE 3 Start point of target End point of target Length oftarget Clone No. sequence sequence sequence (bp) 1 1 19 19 2 1 20 20 3 122 22 4 2 31 30 5 5 30 26 6 29 53 25 7 35 60 26 8 35 62 28 9 76 103 2810 78 105 28 11 83 112 30 12 87 112 26 13 95 120 26 14 96 120 25 15 97121 25 16 109 133 25 17 121 146 26 18 144 170 27 19 148 174 27 20 150174 25 21 175 200 26 22 216 242 27 23 221 260 40 24 230 256 27 25 245267 23 26 268 296 29 27 275 300 26 28 276 306 31 29 278 308 31 30 279306 28 31 283 309 27 32 301 326 26 33 302 328 27 34 330 361 32 35 334359 26 36 372 398 27 37 401 428 28 38 534 563 30 39 534 566 33 40 536563 28 41 539 565 27 42 543 567 25 43 543 570 28 44 545 569 25 45 561589 29 46 567 589 23 47 603 629 27 48 608 640 33 49 642 660 19 50 642663 22 51 642 670 29 52 650 679 30 53 663 689 27 54 682 708 27 55 710736 27 56 711 741 31 57 713 740 28 58 774 801 28 59 789 816 28 60 802836 35 61 824 850 27 62 824 852 29 63 824 854 31 64 824 857 34 65 827858 32 66 828 853 26 67 834 858 25 68 834 858 25 69 834 860 27 70 880906 27 71 886 913 28 72 898 926 29 73 900 922 23 74 905 930 26 75 907934 28 76 912 937 26 77 917 946 30 78 932 952 21 79 950 968 19 80 9861013 28 81 990 1019 30 82 1015 1042 28 83 1022 1049 28 84 1046 1071 2685 1062 1089 28 86 1073 1102 30 87 1095 1124 30 88 1112 1137 26 89 11221145 24 90 1138 1169 32 91 1149 1174 26 92 1149 1182 34 93 1150 1181 3294 1157 1181 25 95 1166 1191 26 96 1180 1207 28 97 1211 1237 27 98 12541278 25 99 1340 1365 26 100 1340 1370 31 101 1416 1445 30 102 1422 144827 103 1425 1453 29 104 1428 1460 33 105 1441 1468 28 106 1451 1480 30107 1463 1491 29 108 1464 1489 26 109 1465 1490 26 110 1498 1517 20 1111498 1517 20 112 1499 1526 28 113 1501 1534 34 114 1502 1529 28 115 15041529 26 116 1504 1530 27 117 1504 1534 31 118 1508 1526 19 119 1532 155726 120 1535 1563 29 121 1555 1578 24 122 1584 1612 29 123 1588 1615 28124 1591 1615 25 125 1591 1619 29 126 1602 1626 25 127 1602 1629 28 1281610 1637 28 129 1613 1637 25 130 1619 1645 27 131 1622 1647 26 132 16801707 28 133 1687 1713 27 134 1729 1746 18 135 1730 1746 17 136 1730 174617 137 1744 1758 15 138 1744 1768 25 139 1744 1773 30 140 1765 1796 32141 1786 1811 26 142 1821 1839 19 143 1821 1842 22 144 1821 1844 24 1451863 1890 28 146 1927 1951 25 147 1940 1965 26 148 1948 1984 37 149 19491976 28 150 1951 1979 29 151 1957 1982 26 152 1957 1982 26 153 1963 198725 154 1963 1989 27 155 1963 1990 28 156 1964 1987 24 157 1965 1990 26158 1974 2000 27 159 1978 2008 31

Among target sequences of 159 clones, RNA sequences corresponding to therepresentative target region are represented by SEQ ID NOs:14 to 23

(3) Search of Mouse, Rat and Human Sequences Homologous to the TargetSequence Contained in the siRNA Expression Unit

Sequences corresponding to the target sequences represented by SEQ IDNOs:14 to 23 obtained in the item (2) were searched in mouse, rat andhuman FUT8 sequences as follows.

SEQ ID NOs:2, 3, and 4 show mouse, rat and human FUT8 sequences,respectively. Among the sequences, sequences corresponding to the targetsequences represented by SEQ ID NOs:14 to 23 obtained in the item (2)were searched. In this search, completely matched sequences wereexcluded.

Each sequence number of the selected sequences is shown below. MouseFUT8 sequence corresponding to SEQ ID NO:14 is represented by SEQ IDNO:85; human FUT8 sequence corresponding to SEQ ID NO:14 is representedby SEQ ID NO: 86; mouse FUT8 sequence corresponding to SEQ ID NO:15 isrepresented by SEQ ID NO:24; human FUT8 sequence corresponding to SEQ IDNO:15 is represented by SEQ ID NO:25; rat FUT8 sequence corresponding toSEQ ID NO:15 is represented by SEQ ID NO:87; human FUT8 sequencecorresponding to SEQ ID NO:16 is represented by SEQ ID NO:26; human,mouse, and rat FUT8 sequences corresponding to SEQ ID NO:17 arerepresented by SEQ ID NO:27; mouse FUT8 sequence corresponding to SEQ IDNO:18 is represented by SEQ ID NO:28; human FUT8 sequence correspondingto SEQ ID NO:18 is represented by SEQ ID NO: 29; rat FUT8 sequencecorresponding to SEQ ID NO:18 is represented by SEQ ID NO:30; mouse andrat FUT8 sequences corresponding to SEQ ID NO:19 are represented by SEQID NO:31; human FUT8 sequence corresponding to SEQ ID NO:19 isrepresented by SEQ ID NO:32; mouse FUT8 sequence corresponding to SEQ IDNO:20 is represented by SEQ ID NO:33; human FUT8 sequence correspondingto SEQ ID NO:20 is represented by SEQ ID NO:34; mouse FUT8 sequencecorresponding to SEQ ID NO:21 is represented by SEQ ID NO:88; human FUT8sequence corresponding to SEQ ID NO:21 is represented by SEQ ID NO:89;and rat FUT8 sequence corresponding to SEQ ID NO:22 is represented bySEQ ID NO:35.

4. Construction of Effective α1,6-Fucosyltransferase (FUT8)-TargetingsiRNA Expression Vector

Among target regions of FUT8-targeting siRNA obtained in the item 3 ofthis Example, siRNA expression vectors which target at the sequencecontained in SEQ ID NO:15 or 16 were constructed according to thefollowing procedure (FIGS. 7, 8, and 9):

Among plasmids containing the siRNA expression cassette obtained in theitem 3(1) of this Example, plasmids were selected, which contain the DNAsequence represented by SEQ ID NO:72 which is a target sequencecontained in SEQ ID NO:15 equivalent to clone No. 31 shown in Table 1 asa sense DNA, and a nucleotide sequence complementary to the DNA sequencerepresented by SEQ ID NO:72 as an antisense DNA (hereinafter referred toas “FUT8shRNA/lib2B/pPUR”), and plasmids containing a DNA sequencecorresponding to SEQ ID NO:16 equivalent to clone No. 72 shown in Table3 as a sense DNA, and a nucleotide sequence complementary to the DNAsequence corresponding to SEQ ID NO:16 as an antisense DNA (hereinafterreferred to as “FUT8shRNA/lib3/pPUR”) (FIG. 7).

First, after 1 μg of FUT8shRNA/lib2B/pPUR or FUT8shRNA/lib3/pPUR plasmidwas dissolved in 30 μL of NEBuffer for EcoRI (manufactured by NewEngland Biolabs) and digested with 10 units of restriction enzymes EcoRIand XhoI (manufactured by New England Biolabs) for 3 hours at 37° C.,the reaction solution was subjected to agarose gel electrophoresis, anda DNA fragment (about 250 bp) containing human tRNA-val promoter-shorthairpin type RNA-terminator sequence expression cassette was recoveredusing RECOCHIP (manufactured by TAKARA BIO).

Also, 1 μg of plasmid pBluescript II KS(+) was dissolved in 30 μL ofNEBuffer for EcoRI (manufactured by New England Biolabs) and digestedwith 10 units of restriction enzymes EcoRI and XhoI (manufactured by NewEngland Biolabs) for 2 hours at 37° C. After the reaction, 13 μL ofsterilized water, 5 μL of 10× alkaline phosphatase buffer, and 1 unit ofalkaline phosphatase E. coli C75 (manufactured by TAKARA BIO) were addedto the reaction solution for carrying out dephosphorylation reaction at37° C. for 1 hour, the reaction solution was subjected to agarose gelelectrophoresis, and an EcoRI-XhoI fragment (about 2.9 Kb) derived fromplasmid pBluescript II KS(+) was recovered using RECOCHIP (manufacturedby TAKARA BIO).

Then, 8 μL of the above EcoRI-XhoI DNA fragment (about 250 bp), 2 μL ofthe EcoRI-XhoI fragment (about 2.9 Kb) derived from plasmid pBluescriptII KS(+), and 10 μL of Ligation High (manufactured by TOYOBO) were mixedand allowed to react for 2 hours at 16° C. E. coli DH5α (manufactured byInvitrogen) was transformed with the reaction solution, and plasmidswere isolated from the resulting ampicillin-resistant clones usingQIAprep spin Mini prep Kit (manufactured by Qiagen). Hereinafter, amongthe above plasmids, plasmids into which DNA fragments (about 250 bp)derived from plasmids FT8libB/pPUR and FT8lib3/pPUR are inserted arereferred to as “FT8libB/pBS” and “FT8lib3/pBS”, respectively.

After 1 μg of plasmid FT8libB/pBS or FT8lib3/pBS was dissolved in 20 μLof NEBuffer 4 (manufactured by New England Biolabs) and digested with 10units of a restriction enzyme SmaI (manufactured by New England Biolabs)at 25° C. for 5 hours, the restriction enzyme SmaI was inactivated byheating at 65° C. for 20 minutes. To the reaction solution, 14.6 μL ofsterilized water, 4 μL of 10× NEBuffer 2 (manufactured by New EnglandBiolabs), 0.4 μL of 100xBSA (manufactured by New England Biolabs), and10 units of a restriction enzyme XhoI (manufactured by New EnglandBiolabs) were added, and digested overnight at 37° C., then the reactionsolution was subjected to on agarose gel electrophoresis, and an DNAfragment containing a human tRNA-val promoter-short hairpin-typeRNA-terminator sequence expression cassette (about 250 bp) was recoveredusing RECOCHIP (manufactured by TAKARA BIO).

Also, 1 μg of plasmid pAGE249 was dissolved in 30 μL of NEBuffer 1(manufactured by New England Biolabs) and digested with 10 units ofrestriction enzymes NaeI and XhoI (manufactured by New England Biolabs)for 6 hours at 37° C. After the digestion reaction, 22 μL of sterilizedwater, 6 μL of 10× alkaline phosphatase buffer, and 1 unit of alkalinephosphatase E. coli C75 (manufactured by TAKARA BIO) were added to thereaction solution for carrying out dephosphorylation reaction at 37° C.for 1 hour. The reaction solution was subjected to agarose gelelectrophoresis, and a NaeI-XhoI fragment (about 4.4 Kb) derived fromplasmid pAGE249 was recovered using RECOCHIP (manufactured by TAKARABIO). Also, pAGE249 is a derivative of pAGE248 [J. Biol. Chem., 269,14730 (1994)], namely a pAGE248 vector from which a 2.7 Kb fragmentcontaining dihydrofolate reductase (dhfr) gene expression unit, digestedwith SphI restriction enzyme, was removed.

Then, 10 μL of the above DNA fragment (about 250 bp), 5 μL of theNaeI-XhoI fragment (about 4.4 Kb) derived from plasmid pAGE249, and 15μL of Ligation High (manufactured by TOYOBO) were mixed and allowed toreact overnight at 16° C. E. coli DH5α (manufactured by Invitrogen) wastransformed with the reaction solution, and plasmid DNA was isolatedfrom the resulting ampicillin-resistant clones using QIAprep spin Miniprep Kit (manufactured by Qiagen). The nucleotide sequence of the DNAinserted in each plasmid was determined with DNA sequencer 377(manufactured by Perkin Elmer) and BigDye Terminator v3.0 Cyclesequencing Kit (manufactured by Applied Biosystems) according to themanufacturer's instruction. pAGE249-seq FW (SEQ ID NO:73) andpAGE249-seq RV (SEQ ID NO:74) were used as sequencing primers, andagreement of inserted DNA sequences with the sequence corresponding tothe EcoRI-XhoI fragment which is a human tRNA-val promoter-shorthairpin-type RNA expression unit contained in plasmidFUT8shRNA/lib2B/pPUR or FUT8shRNA/lib3/pPUR was confirmed. Hereinafter,among the above plasmids, plasmids into which DNA fragments (about 250bp) derived from plasmids FUT8shRNA/lib2B/pPUR and FUT8shRNA/lib3/pPURare inserted are referred to as “FT8libB/pAGE” and “FT8lib3/pAGE”,respectively.

EXAMPLE 5

Preparation of Lectin-Resistant CHO/DG44 Cell in the Presence ofL-Fucose by Co-Introducing FUT8-Targeting siRNA Expression Plasmid intoLectin-Resistant Clone Introduced with GMD-Targeting siRNA ExpressionPlasmid:

1. Obtaining and Culturing of Lectin-Resistant Clone in the Presence ofL-Fucose by Introducing FUT8-Targeting siRNA Expression Vector intoLectin-Resistant Clone Introduced with GMD-Targeting siRNA ExpressionVector

(1) Obtaining of Lectin-Resistant Clone in the Presence of L-Fucose byIntroducing FUT8-Targeting siRNA Expression Vector

Lectin-resistant clones were obtained under L-fucose-added culturingconditions by introducing FT8libB/pAGE or FT8lib3/pAGE, theFUT8-targeting siRNA expression vector constructed in Example 2, intolectin-resistant clone 12-GMDB-2 or 12-GMDB-5 introduced with theGMD-targeting siRNA expression vector obtained in Example 1.

Transfection of plasmid FT8libB/pAGE or FT8lib3/pAGE into clone12-GMDB-2 or 12-GMDB-5 was carried out by the same method as in the item2(1) of Example 1.

After the transfection, the cell suspension was suspended in a basalmedium containing 12 μg/mL puromycin (manufactured by SIGMA) andinoculated into four 10 cm-culture dishes for adherent cells(manufactured by Falcon). After culturing under conditions of 37° C. and5% CO₂ for 24 hours, the culture supernatant was removed, and a basalmedium containing 400 μg/mL hygromycin (manufactured by WAKO) and 3μg/mL puromycin (manufactured by SIGMA) was added thereto, followed byculturing for further 8 days. Subsequently, culture supernatant wasremoved from one of the dishes, a basal medium containing 400 μg/mLhygromycin (manufactured by WAKO) and 3 μg/mL puromycin (manufactured bySIGMA) was added thereto, followed by culturing for further 6 to 8 days,and the appeared hygromycin-resistant colonies were counted. Also, theculture supernatant was removed from the remaining dishes, and a basalmedium containing 400 μg/mL hygromycin (manufactured by WAKO), 3 μg/mLpuromycin (manufactured by SIGMA), 100 μmol/L L-fucose (manufactured byNacalai Tesque), and 0.5 mg/mL LCA (manufactured by VECTOR) was addedthereto, followed by culturing for further 9 days, and lectin-resistantclones in the presence of 100 μmol/L L-fucose, namely lectin-resistantclones co-introduced with FUT8-targeting siRNA expression vector andGMD-targeting siRNA expression vector, were obtained.

(2) Expansion Culture of Lectin-Resistant Clones

Lectin-resistant clones obtained in the item (1) by co-introducingFUT8-targeting siRNA expression vector and GMD-targeting siRNAexpression vector were expansion cultured according to the followingprocedure.

First, the number of colonies appeared in each dish was counted. Then,lectin-resistant colonies were scraped and sucked up with a pipetteman(manufactured by GILSON) under observation with stereoscopic microscope,and collected into a U-shaped-bottom 96-well plate for adherent cells(manufactured by ASAHI TECHNOGLASS). After trypsin treatment, each clonewas inoculated into a flat-bottom 96-well plate for adherent cells(manufactured by Greiner), and cultured in a basal medium containing 400μg/mL hygromycin (manufactured by WAKO) and 3 μg/mL puromycin(manufactured by SIGMA) under conditions of 5% CO₂ and 37° C. overnight.The culture supernatant was removed after the culturing, and the cellswere cultured in a basal medium containing 400 μg/mL hygromycin(manufactured by WAKO), 3 μg/mL puromycin (manufactured by SIGMA), 100μmol/L L-fucose (manufactured by Nacalai Tesque) and 0.5 mg/mL LCA(manufactured by VECTOR) for further 6 days. After the culturing, eachclone of the above plate was expansion cultured in a basal mediumcontaining 400 μg/mL hygromycin (manufactured by WAKO) and 3 μg/mLpuromycin (manufactured by SIGMA). Hereinafter, among clones used in theexpansion culture, lectin-resistant clones obtained by introducingFT8libB/pAGE into clone 12-GMDB-2 are referred to as “12-GB2/FB-1”,“12-GB2/FB-2”, “12-GB2/FB-3”, “12-GB2/FB-4” and “12-GB2/FB-5”; those byintroducing FT8lib3/pAGE into clone 12-GMDB-2 are referred to as“12-GB2/F3-1”, “12-GB2/F3-2”, “12-GB2/F3-3”, “12-GB2/F3-4” and“12-GB2/F3-5”; those by introducing FT8libB/pAGE into clone 12-GMDB-5are referred to as “12-GB5/FB-1”, “12-GB5/FB-2”, “12-GB5/FB-3”,“12-GB5/FB-4” and “12-GB5/FB-5”; those by introducing FT8lib3/pAGE intoclone 12-GMDB-5 are referred to as “12-GB5/F3-1”, “12-GB5/F3-2”,“12-GB5/F3-3”, “12-GB5/F3-4” and “12-GB5/F3-5”, respectively. Also,clones 12-GB5/FB-1 and 12-GB5/F3-2 have been deposited to InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology (Tsukuba Central 6, 1, Higashi 1-chome,Tsukuba-shi, Ibaraki-ken, Japan n) as FERM BP-10049 and FERM BP-10050,respectively, on Jul. 1, 2004.

2. Determination of the Amount of GMD mRNA and FUT8 mRNA inLectin-Resistant Clones into which GMD-Targeting Expression Vector andFUT8-Targeting siRNA Expression Vector were Introduced in the Presenceof L-Fucose

(1) Preparation of Total RNA

A total RNA was prepared from the parent clone 32-05-12 before vectorintroduction, lectin-resistant clones 12-GMDB-2 and 12-GMDB-5 into whichthe GMD-targeting siRNA expression vector was introduced obtained inExample 1, and lectin-resistant clones into which the GMD-targetingexpression vector and the FUT8-targeting siRNA expression vector wereco-introduced obtained in the item 1 of this Example according to thefollowing procedure.

Clone 32-05-12 was suspended in a basal medium, clones 12-GMDB-2 and12-GMDB-5 were suspended in basal medium supplemented with 12 μg/mLpuromycin (manufactured by SIGMA), and lectin-resistant clones intowhich FUT8-targeting siRNA expression vector and GMD-targeting siRNAexpression vector were introduced were suspended in a basal mediumcontaining 400 μg/mL hygromycin (manufactured by WAKO) and 3 μg/mLpuromycin (manufactured by SIGMA), at a density of 3×10⁵ cells/mL, and 4mL each was inoculated into 6 cm-dishes for adherent cells (manufacturedby Falcon). The cells were cultured under conditions of 5% CO₂ and 37°C. for 3 days, and each cell suspension was recovered by trypsintreatment and centrifugation at 1,000 rpm at 4° C. for 5 minutes. Afterthe recovered cells were suspended in Dulbecco's PBS buffer(manufactured by Invitrogen), cells were again recovered byre-centrifugation at 1,000 rpm at 4° C. for 5 minutes, and a total RNAwas extracted using RNeasy (manufactured by QIAGEN). The method wascarried out according to the manufacturer's instruction, and theprepared total RNA was dissolved in 40 μL of sterilized water.

(2) Synthesis of Single-Stranded cDNA

A single-stranded cDNA was synthesized from 3 μg of each of the totalRNA obtained in the item (1) by reverse transcription reaction witholigo (dT) primer in 20 μL reaction system using SUPERSCRIPT™Preamplification System for First Strand cDNA Synthesis (manufactured byInvitrogen) according to the manufacturer's instruction. Subsequently,the reaction solution was treated with RNase and the final reactionvolume was adjusted to 40 μL. In addition, each of the reactionsolutions was diluted 50-fold with sterilized water, and used fordetermination of the amount of gene transcription described below.

(3) Determination of the Amount of GMD Gene Transcription by SYBR-PCR

The amount of mRNA transcribed from GMD gene and β-actin gene werequantified as described below. Also, plasmid pAGE249GMD containing CHOcell-derived GMD cDNA described in Example 15 of WO02/31140, eachdiluted at concentration of 0.0512 fg/μL, 0.256 fg/μL, 1.28 fg/μL, 6.4fg/μL, 32 fg/μL and 160 fg/μL, were used as internal controls of GMDdetermination, and β-actin standard plasmid described in Example 9 ofWO02/31140, each diluted at concentration of 1.28 fg/μL, 6.4 fg/μL, 32fg/μL, 160 fg/μL, 800 fg/μL and 4,000 fg/μL, were used as internalcontrols of β-actin determination. Furthermore, as PCR primers, forwardand reverse primers represented by SEQ ID NOs:64 and 65, respectively,were used to amplify GMD, and forward and reverse primers represented bySEQ ID NOs:66 and 67, respectively, were used to amplify β-actin.

Then, 20 μL of reaction solution [R-PCR buffer (manufactured by TAKARABIO), 2.5 mmol/L Mg²⁺ Solution for R-PCR (manufactured by TAKARA BIO),0.3 mmol/L dNTP mixture (manufactured by TAKARA BIO), 0.3 μmol/L forwardprimer, 0.3 μmol/L reverse primer, 2×10⁻⁵ diluted SYBR GreenI, 1 unitTaKaRa Ex Taq R-PCR] containing 5 μL of the single-stranded cDNAsolution prepared in the item (2) or each concentration of internalcontrol plasmid solution was prepared using For Real Time PCR TaKaRa ExTaq R-PCR Version (manufactured by TAKARA BIO). The prepared reactionsolution was dispensed into each well of a 96-well Polypropylene PCRPlate (manufactured by Falcon), and the plate was sealed with PlateSealer (manufactured by Edge Biosystems). ABI PRISM 7700 SequenceDetection System was used for PCR and analysis, and the amount of GMDmRNA and the amount of β-actin mRNA were determined according to themanufacturer's instruction.

A calibration curve was obtained based upon the measurements with theinternal control plasmid, and the amount of GMD mRNA and the amount ofβ-actin mRNA were converted into numerical terms. In addition, assumingthat the amount of mRNA transcribed from β-actin gene are uniform amongthe clones, the relative amount of GMD mRNA to the amount of β-actinmRNA were calculated and compared, and the results are shown in FIG. 10.The amount of GMD mRNA in all the clones obtained by co-introducingFUT8-targeting siRNA expression vector and GMD-targeting siRNAexpression vector were reduced to about 10% in comparison with that inthe parent clone 32-05-12.

(4) Determination of the Amount of FUT8 Gene Transcription by SYBR-PCR

The amount mRNA transcribed from FUT8 gene and β-actin gene weredetermined as described below. Also, FUT8 standard plasmid described inExample 9 of WO02/31140, each diluted at concentration of 0.0512 fg/μL,0.256 fg/μL, 1.28 fg/μL, 6.4 fg/μL, 32 fg/μL and 160 fg/μL, were used asinternal controls of FUT8 determination; β-actin standard plasmiddescribed in Example 9 of WO02/31140, each diluted at concentration of1.28 fg/μL, 6.4 fg/μL, 32 fg/μL, 160 fg/μL, 800 fg/μL and 4,000 fg/μL,were used as internal controls of β-actin determination. Furthermore, asPCR primer, forward and reverse primers represented by SEQ ID NOs:75 and76, respectively, were used to amplify FUT8, and forward and reverseprimers represented by SEQ ID NOs:66 and 67, respectively, were used toamplify β-actin.

Then, 20 μL of reaction solution [R-PCR buffer (manufactured by TAKARABIO), 2.5 mmol/L Mg²⁺ Solution for R-PCR (manufactured by TAKARA BIO),0.3 mmol/L dNTP mixture (manufactured by TAKARA BIO), 0.3 μmol/L forwardprimer, 0.3 μmol/L reverse primer, 2×10⁻⁵ diluted SYBR GreenI, and 1unit TaKaRa Ex Taq R-PCR] containing 5 μL of the single-stranded cDNAsolution prepared in the item (2) or each concentration of internalcontrol plasmid solution was prepared using For Real Time PCR TaKaRa ExTaq R-PCR Version (manufactured by TAKARA BIO). The prepared reactionsolution was dispensed into each well of a 96-well Polypropylene PCRPlate (manufactured by Falcon), and the plate was sealed with PlateSealer (manufactured by Edge Biosystems). ABI PRISM 7700 SequenceDetection System was used for PCR and analysis, and the amount of GMDmRNA and the amount of β-actin mRNA were determined according to themanufacturer's instruction.

A calibration curve was obtained based upon the measurements with theinternal control plasmid, and the amount of FUT8 mRNA and the amount ofβ-actin mRNA were converted into numerical terms. In addition, assumingthat the amount of mRNA transcribed from β-actin gene are uniform amongthe clones, the relative amount of FUT8 mRNA to the amount of β-actinmRNA were calculated and compared, and the results are shown in FIG. 11.The amounts of FUT8 mRNA in all the clones obtained by co-introducingFUT8-targeting siRNA expression vector and GMD-targeting siRNAexpression vector were reduced to about 10% in comparison with that inthe parent clone 32-05-12.

EXAMPLE 6

Production of Antibody Compositions using CHO/DG44 Cell into whichGMD-Targeting siRNA Expression Plasmid and FUT8-Targeting siRNAExpression Plasmid were Introduced:

1. Obtaining of Antibody Compositions Under Culturing in the Absence ofL-Fucose

Anti-CCR4 chimeric antibody compositions produced by the parent clone32-05-12 before vector introduction, lectin-resistant clones 12-GMDB-2and 12-GMDB-5 into which GMD-targeting siRNA expression plasmid wasintroduced obtained in Example 1, and lectin-resistant clones12-GB2/FB-1,12-GB2/FB-3,12-GB2/F3-3,12-GB2/F3-5,12-GB5/FB-1,12-GB5/FB-2,12-GB5/F3-2 and 12-GB5/F3-4 into whichFUT8-targeting siRNA expression vector and GMD-targeting siRNAexpression vector were co-introduced obtained in Example 3, wereobtained according to the following procedure.

Clone 32-05-12 was suspended in a basal medium, clones 12-GMDB-2 and12-GMDB-5 were suspended in a basal medium containing 12 μg/mL puromycin(manufactured by SIGMA), and clones 12-GB2/FB-1, 12-GB2/FB-3,12-GB2/F3-3, 12-GB2/F3-5,12-GB5/FB-1,12-GB5/FB-2,12-GB5/F3-2 and12-GB5/F3-4 were suspended in a basal medium containing 400 μg/mLhygromycin (manufactured by WAKO) and 3 μg/mL puromycin (manufactured bySIGMA), at a density of 3×10⁵ cells/mL, and were inoculated at 15 mLinto a T75 flask for adherent cells (manufactured by Greiner). Afterculturing under conditions of 5% CO₂ and 37° C. for 6 days, the culturesupernatant was removed, and after washing with 10 mL of Dulbecco's PBS(manufactured by Invitrogen), 20 mL of EXCELL301 medium (manufactured byJRH Bioscience) was added. After culturing under conditions of 5% CO₂and 37° C. for 7 days, the culture supernatant was recovered, andanti-CCR4 chimeric antibody compositions were purified using a MabSelectcolumn (manufactured by Amersham Bioscience) according to themanufacturer's instruction. After exchange into 10 mmol/L KH₂PO₄ bufferusing Econo-Pac 10DG (manufactured by Bio Rad), anti-CCR4 chimericantibody compositions purified from culture supernatants of variousclones were subjected to steric filtration by using Millex GV(manufactured by MILLIPORE) of 0.22 μm pore size.

2. Obtaining of Antibody Compositions Under Culturing in the Presence ofL-Fucose

Anti-CCR4 chimeric antibody compositions produced under presence ofL-fucose culturing conditions by the parent clone 32-05-12 before vectorintroduction, lectin-resistant clones 12-GMDB-2 and 12-GMDB-5 into whichthe GMD-targeting siRNA expression plasmid was introduced obtained inExample 1, and lectin-resistant clones 12-GB2/FB-1, 12-GB2/FB-3,12-GB2/F3-3, 12-GB2/F3-5, 12-GB5/FB-1, 12-GB5/FB-2, 12-GB5/F3-2 and12-GB5/F3-4 into which the GMD-targeting siRNA expression plasmid andthe FUT8-targeting siRNA expression plasmid were introduced obtained inExample 3, were obtained according to the following procedure:

Clone 32-05-12 was cultured in a basal medium, clones 12-GMDB-2 and12-GMDB-5 were suspended in a basal medium supplemented with 12 μg/mLpuromycin (manufactured by SIGMA), and clones 12-GB2/FB-1, 12-GB2/FB-3,12-GB2/F3-3, 12-GB2/F3-5, 12-GB5/FB-1, 12-GB5/FB-2, 12-GB5/F3-2 and12-GB5/F3-4 were suspended in a basal medium supplemented with 400 μg/mLhygromycin (manufactured by WAKO) and 3 μg/mL puromycin (manufactured bySIGMA), at a density of 3×10⁵ cells/mL, and were inoculated at 15 mLinto a T75 flask for adherent cells (manufactured by Greiner). Afterculturing under conditions of 5% CO₂ and 37° C. for 3 days, the culturesupernatant was removed, and the culture was replaced by a basal mediumsupplemented with 500 μmol/L L-fucose (manufactured by Nacalai Tesque)for clone 32-05-12, a basal medium supplemented with 500 μmol/L L-fucose(manufactured by Nacalai Tesque) and 12 μg/mL puromycin (manufactured bySIGMA) for clones 12-GMDB-2 and 12-GMDB-5, and a basal mediumsupplemented with 500 μmol/L L-fucose (manufactured by Nacalai Tesque),400 μg/mL hygromycin (manufactured by WAKO), and 3 μg/mL puromycin(manufactured by SIGMA) for clones 12-GB2/FB-1, 12-GB2/FB-3,12-GB2/F3-3, 12-GB2/F3-5, 12-GB5/FB-1, 12-GB5/FB-2, 12-GB5/F3-2 and12-GB5/F3-4. After culturing under conditions of 5% CO₂ and 37° C. forfurther 3 days, the culture supernatant was removed, and after washingwith 10 mL of Dulbecco's PBS (manufactured by Invitrogen), 20 mL ofEXCELL301 medium (manufactured by JRH Bioscience) supplemented with 500μmol/L L-fucose (manufactured by Nacalai Tesque) was added. Afterculturing under conditions of 5% CO₂ and 37° C. for 3 days, 1 mL of 10mmol/L L-fucose (manufactured by Nacalai Tesque) was added to eachflask, followed by culturing for further 4 days. After the culturing,the culture supernatant was recovered, and anti-CCR4 chimeric antibodieswere purified using a MabSelect column (manufactured by AmershamBioscience) according to the manufacturer's instruction. After exchangewith 10 mmol/L KH₂PO₄ buffer using Econo-Pac 10DG (manufactured by BioRad), anti-CCR4 chimeric antibody compositions purified from culturesupernatant of various clones were subjected to steric filtration byusing Millex GV (manufactured by MILLIPORE) 0.22 μm pore size.

3. Composition Analysis of Monosaccharide of Antibody Compositions

Composition analysis of monosaccharide was carried out for eachanti-CCR4 chimeric antibody compositions obtained in the items 1 and 2of this Example according to the known method [Journal of LiquidChromatography, 6, 1577 (1983)]. TABLE 4 Fucose(−) % ofanti-CCR4-chimeric antibody composition produced by each clone Fucose(−)% Culturing in the presence Culturing in the absence of Clone ofL-fucose L-fucose 32-05-12  6%  3% 12-GMDB-2  6% 78% 12-GB2/FB-1 66% 94%12-GB2/FB-3 67% 92% 12-GB2/F3-3 81% 98% 12-GB2/F3-5 79% 97% 12-GMDB-5 8% 79% 12-GB5/FB-1 71% 97% 12-GB5/FB-2 76% 96% 12-GB5/F3-2 81% 95%12-GB5/F3-4 67% 91%

The ratios of antibody having sugar chains in which fucose is not boundto N-acetylglucosamine in the reducing end in the complex typeN-glycoside-linked sugar chain calculated from the monosaccharidecomposition ratio of each antibody composition (hereinafter referred toas “fucose(−)% of antibody composition”) are shown in Table 4.

Under culturing in the absence of L-fucose, fucose(−)% of the antibodycomposition produced from the parent clone 32-05-12 used forintroduction of the GMD-targeting siRNA expression vector was 3%, whilethose of lectin-resistant clones 12-GMDB-2 and 12-GMDB-5 into whichGMD-targeting siRNA was introduced were 78% and 79%, respectively,demonstrating significantly increased fucose(−)% of the antibodycompositions in comparison with that of the parent cell. In addition,fucose(−)% of the antibody compositions produced by lectin-resistantclones 12-GB2/FB-1, 12-GB2/FB-3, 12-GB2/F3-3, 12-GB2/F3-5, 12-GB5/FB-1,12-GB5/FB-2, 12-GB5/F3-2 and 12-GB5/F3-4 into which GMD-targeting siRNAand FUT8-targeting siRNA were co-introduced were 91 to 98%,demonstrating further increase in fucose(−)% of antibody compositions incomparison with those of lectin-resistant clones introduced withGMD-targeting siRNA alone.

Also, under culturing in the presence of L-fucose, fucose(−)% ofantibody composition produced by the parent clone 32-05-12 used forintroduction of the GMD-targeting siRNA expression vector was 6%, whilethose of lectin-resistant clones 12-GMDB-2 and 12-GMDB-5 into whichGMD-targeting siRNA was introduced were 6% and 8%, respectively. On theother hand, fucose(−)% of the antibody compositions produced bylectin-resistant clones 12-GB2/FB-1, 12-GB2/FB-3, 12-GB2/F3-3,12-GB2/F3-5, 12-GB5/FB-1, 12-GB5/FB-2, 12-GB5/F3-2 and 12-GB5/F3-4 intowhich GMD-targeting siRNA and FUT8-targeting siRNA were co-introducedwere values between 66 to 81%, demonstrating a significant increase infucose(−)% of the antibody compositions in comparison with those oflectin-resistant clones into which GMD-targeting siRNA was introducedalone.

As described above, fucose(−)% of the antibody compositions produced bylectin-resistant clones co-introduced with GMD-targeting andFUT8-targeting siRNA were higher than that of lectin-resistant clonesinto which GMD-targeting siRNA was introduced alone under culturing inthe absence or presence of L-fucose, therefore, regardless of thepresence or absence of L-fucose, effect of inhibiting the addition ofα1,6-fucose to complex sugar chains of cell-produced antibodycompositions based on co-introducing GMD-targeting siRNA andFUT8-targeting siRNA were higher than those of introducing GMD-targetingsiRNA alone.

In addition, fucose(−)% of the antibody compositions produced bylectin-resistant clones into which GMD-targeting siRNA andFUT8-targeting siRNA were co-introduced were higher in culturing in theabsence of L-fucose than culturing in the presence of L-fucose whereeffect of rising fucose(−)% of GMD-targeting siRNA are lost, it is shownthat the suppressive effects on addition of α1,6-fucose to complex sugarchains of cell-produced antibody compositions by co-introducingGMD-targeting siRNA and FUT8-targeting siRNA is higher than thesuppressive effect of introducing GMD-targeting siRNA alone.

EXAMPLE 7

Serum-Free Fed-Batch Culture of CHO/DG44 Cell into which GMD-TargetingExpression Vector and FUT8-Targeting siRNA Expression Vector wereIntroduced:

1. Adaptation of CHO/DG44 Cell into which GMD-Targeting ExpressionVector and FUT8-Targeting siRNA Expression Vector were Introduced toSerum-Free Medium

The parent clone 32-05-12 before vector introduction, andlectin-resistant clones 12-GB2/FB-1, 12-GB2/FB-3, 12-GB2/F3-3,12-GB2/F3-5, 12-GB5/FB-1, 12-GB5/FB-2, 12-GB5/F3-2 and 12-GB5/F3-4 intowhich the GMD-targeting siRNA expression vector and FUT8-targeting siRNAexpression vector were introduced obtained in Example 3, were adapted toa serum-free medium according to the following procedure.

Clone 32-05-12 was suspended in a basal medium, and clones 12-GB2/FB-1,12-GB2/FB-3, 12-GB2/F3-3, 12-GB2/F3-5, 12-GB5/FB-1, 12-GB5/FB-2,12-GB5/F3-2 and 12-GB5/F3-4 were suspended in a basal mediumsupplemented with 400 μg/mL hygromycin (manufactured by WAKO) and 3μg/mL puromycin (manufactured by SIGMA), at a density of 3×10⁵ cells/mL,and each was inoculated at 15 mL into a T75 flask for adherent cells(manufactured by Greiner). After culturing under conditions of 5% CO₂and 37° C. for 3 days, cells were suspended by trypsin treatment, andcells were recovered by centrifugation at 1,000 rpm for 5 minutes. Clone32-05-12 was suspended in EX-CELL302 medium (manufactured by JRH)containing 500 nmol/L MTX (manufactured by SIGMA), 6 mmol/L L-glutamine(manufactured by Invitrogen), 50 μg/mL gentamicin (manufactured byNacalai Tesque), and 100 nmol/L 3,3,5-Triiodo-L-thyronine (manufacturedby SIGMA) (hereinafter referred to as “serum-free medium”), and clones12-GB2/FB-1, 12-GB2/FB-3, 12-GB2/F3-3, 12-GB2/F3-5, 12-GB5/FB-1,12-GB5/FB-2, 12-GB5/F3-2 and 12-GB5/F3-4 were suspended in a serum-freemedium supplemented with 400 μg/mL hygromycin (manufactured by WAKO) and3 μg/mL puromycin (manufactured by SIGMA), at a density of 5×10⁵cells/mL, and 15 mL of the cell suspension was inoculated into a 125 mLconical flask (manufactured by Corning). After ventilating the flaskwith 5% CO₂ (at least 4-fold volume of culture vessel) and sealing theflask, suspension rotation culture was carried out at 90 to 100 rpm and35° C. Passage was repeated at 3 to 4 day intervals, and finally, cloneswhich could grow in the serum-free medium were obtained. Hereinafter,clone 32-05-12 adapted to the serum-free medium is referred to as“32-05-12AF”; clone 12-GB2/FB-1 adapted to the serum-free medium isreferred to as “clone Wi2B-1AF”; clone 12-GB2/FB-3 adapted to theserum-free medium is referred to as “Wi2B-3AF”; clone 12-GB2/F3-3adapted to the serum-free medium is referred to as “Wi23-3AF”; clone12-GB2/F3-5 adapted to the serum-free medium is referred to as“Wi23-5AF”; clone 12-GB5/FB-1 adapted to the serum-free medium isreferred to as “Wi5B-1AF”; clone 12-GB5/FB-2 adapted to the serum-freemedium is referred to as “Wi5B-2AF”; clone 12-GB5/F3-2 adapted to theserum-free medium is referred to as “Wi53-2AF”; and clone 12-GB5/F3-4adapted to serum-free medium is referred to as “Wi53-4AF”, respectively.

2. Serum-Free Fed-Batch Culture Using CHO/DG44 Cell into whichGMD-Targeting Expression Vector and FUT8-Targeting siRNA ExpressionVector were Introduced and Adapted to Serum-Free Medium

Serum-free fed-batch culture was carried out with clones, 32-05-12AF,Wi23-3AF, Wi23-5AF and Wi5B-1AF, adapted to the serum-free medium in theitem 1 of this Example according to the following procedure:

A serum-free fed-batch medium and a feed medium were used for thefed-batch culture.

Clones 32-05-12AF, Wi23-3AF, Wi23-5AF and Wi5B-1AF were suspended in aserum-free fed-batch medium at a density of 3×10⁵ cells/mL, and 40 mLeach of the cell suspension was inoculated into a 250 mL conical flask(manufactured by Corning). After ventilating the flask with 5% CO₂ (atleast 4-fold volume of culture vessel) and sealing the flask, suspensionrotation culture was carried out at 90 to 100 rpm and 35° C. On days 3,6, 9 and 12 after starting the culture, 3.3 mL of a feed medium wasadded to supplement the consumption of amino acids and the like, and 20%(w/v) glucose solution was added at a final concentration of 5,000 mg/Lto adjust the glucose concentration. On days 0, 3, 6, 9, 12 and 14 afterstarting the culture, 2 to 4 mL each culture was collected, and theviable cell number and viability were measured by trypan blue staining,and antibody concentrations contained in each culture supernatant weremeasured by ELISA described in the item 3(1) of this Example. Viablecell number and antibody composition concentration in culturesupernatant at each point of time after starting the culture of clones32-05-12AF and Wi23-5AF are shown in FIGS. 12 and 13, respectively.Viable cell numbers and antibody composition concentrations in culturesupernatants at each point of time after starting the culture weresimilar between clones 32-05-12AF and Wi23-5AF. Therefore, it wasdemonstrated that the target sequences of GMD-targeting siRNA andFUT8-targeting siRNA had no significant effects on the cell growth andantibody production.

3. Determination of Antibodies Having Sugar Chains in which 1-Positionof Fucose is not Bound to 6-Position of N-Acetylglucosamine in theReducing end Through α-Bond Using the Binding Activity to Soluble HumanFcγRIIIa as an Indicator

Fucose(−)% of anti-CCR4 chimeric antibody compositions contained in theserum-free fed-batch culture supernatant of clones 32-05-12AF, Wi23-3AF,Wi23-5AF and Wi5B-1AF obtained in the item 2 of this Example weremeasured using the binding activity to soluble human FcγRIIIa(hereinafter referred to as “shFcγRIIIa”) described in Example 10 ofWO03/85119 as an indicator according to the following procedure.

(1) Determination of Antibody Concentration by ELISA

Antibody concentrations in the culture supernatant were determined inthe same manner as in the item 2(3) of Example 3.

Antibody concentrations of each diluted sample were calculated using thelinear area of the sigmoid curve of the calibration curve prepared witha standard of purified antibody preparation, and each antibodyconcentration of culture supernatants was calculated by multiplyingantibody concentration of the obtained diluted samples by the dilutionrate.

(2) Preparation of Standards with Different Fucose(−)%

Standards with different fucose(−)% were prepared using anti-CCR4chimeric antibodies, YB2/0 cell-derived KM2760-1 and CHO/DG44cell-derived KM3060, described in Example 4 of WO03/85119. Fucose(−)%was measured by composition analysis of monosaccharide described in theitem 3 of Example 4 for a total of 11 standard samples includingKM2760-1, KM3060, and 9 standard samples prepared by mixing KM2760-1 andKM3060; KM2760-1 was 90%; KM3060 was 10%; 9 standard preparation samplesprepared were 82%, 74%, 66%, 58%, 50%, 42%, 34%, 26% and 18%,respectively.

(3) Evaluation of the Binding Activity of Antibody to shFcγRIIIa

BSA conjugate with human CCR4 cell extracellular peptides having theamino acid sequence represented by SEQ ID NO:77 to which an anti-CCR4chimeric antibody reacts was prepared in the same manner as the methoddescribed in the item 2 of Example 4 of WO03/85119.

After 50 μL/well of the prepared BSA conjugate with human CCR4extracellular peptide was dispensed onto 96-well ELISA plates(manufactured by Greiner) at a concentration of 1 μg/mL, the mixture wasleft overnight at 4° C. to adsorb. After washing with PBS, 100 μL/wellof BSA-PBS was added, and was allowed to react for 1 hour at roomtemperature to block remaining active groups. After washing each wellwith Tween-PBS, 50 μL/well of each anti-CCR4 chimeric antibody samplediluted with BSA-PBS was added at 2.5 μg/mL according to the antibodyconcentrations measured by ELISA described in the item (1), and wasallowed to react for 1 hour at room temperature. After washing each wellwith Tween-PBS, 50 μL/well of shFcγRIIIa solution diluted at 5 μg/mLwith BSA-PBS was added thereto, and was allowed to react for 1 hour atroom temperature. After washing each well with Tween-PBS, 50 μL/well ofHRP-labeled mouse antibody Penta-His HRP Conjugate (manufactured byQIAGEN) prepared with BSA-PBS at 0.1 μg/mL was added, and the mixturewas allowed to react for 1 hour at room temperature. After washing withTween-PBS, 50 μL/well of ABTS substrate solution was added, and aftercolor development in the same manner as the method in the item (2),absorbance at 490 nm was measured with absorbance at 415 nm asreference, using a microplate reader.

Binding activities of each anti-CCR4 chimeric antibody standardpreparation prepared in the item (2) with known fucose(−)% to shFcγRIIIaare shown in FIG. 14. Binding activity to shFcγRIIIa increased inproportion to fucose(−)%, and a calibration curve was obtained based onit. Also, in the measurement, calibration curves were made for each96-well ELISA plate.

From the absorbance at 490 nm at which each anti-CCR4 chimeric antibodycomposition contained in the serum-free fed-batch culture supernatantobtained in the item 2 of this Example showed in binding activities toshFcγRIIIa, fucose(−)% of the anti-CCR4 chimeric antibody compositioncontained in culture supernatant was obtained using the calibrationcurve. Results of clones 32-05-12AF and Wi23-5AF are shown in FIG. 15.

Antibody composition contained in the culture supernatant of 32-05-12AFexhibited low binding activity to shFcγRIIIa in the culture, and itsfucose(−)% was approximately 10%. Also, antibody composition containedin the culture supernatant of clones Wi23-3AF, Wi23-5AF, and Wi5B-1AFexhibited high binding activity to shFcγRIIIa in the culture, and theirfucose(−)% were 75 to 90% or higher. Therefore, it was demonstrated thatthe target sequences of GMD-targeting siRNA and FUT8-targeting siRNAcould stably produce antibody compositions with high fucose(−)% withoutaffecting cell growth and antibody production.

4. Sugar Chain Structural Analysis of Antibody Composition Produced atthe End of Serum-Free Fed-Batch Culture

(1) Obtaining of Purified Antibody upon Completion of Serum-FreeFed-Batch Culture

Each anti-CCR4 chimeric antibody composition was purified from culturesupernatants of serum-free fed-batch culture on day 14 of clones32-05-12AF, Wi23-3AF, Wi23-5AF, and Wi5B-1AF obtained in the item 2 ofthis Example, using a MabSelect column (manufactured by AmershamBiosciences) according to the manufacturer's instruction. After exchangewith 10 mmol/L KH₂PO₄ buffer using Econo-Pac 10DG (manufactured by BioRad), each purified anti-CCR4 chimeric antibody composition wassubjected to sterile filtration by using Millex GV (manufactured byMILLIPORE) of 0.22 μm pore size.

(2) Composition Analysis of Monosaccharide of Antibody Compositions

Composition analysis of Monosaccharide was carried out with eachanti-CCR4 chimeric antibody compositions obtained from culturesupernatants of clones adapted to serum-free medium in the item (1)according to the known method [Journal of Liquid Chromatography, 6, 1577(1983)].

Fucose(−)% calculated from composition ratio of monosaccharide of eachantibody composition is shown in Table 5. TABLE 5 Fucose(−) % ofanti-CCR4-chimeric antibody composition produced by each clone CloneFucose(−) % 32-05-12AF  7% Wi23-3AF 87% Wi23-5AF 81% Wi5B-1AF 81%

Fucose(−)% of antibody composition produced by clone 32-05-12AF was 7%,while those of lectin-resistant clones Wi23-3AF, Wi23-5AF and Wi5B-1AFinto which the GMD-targeting siRNA expression vector and theFUT8-targeting siRNA expression vector were co-introduced wereapproximately 80 to 90%, and the comparison with the parent clonedemonstrated that inhibitory effects on α1,6-fucose addition to complexsugar chains of antibody compositions were maintained inlectin-resistant clones into which the GMD-targeting expression vectorand the FUT8-targeting siRNA expression vector were co-introduced evenat the end of the culture.

EXAMPLE 8

Production of Antibody Compositions Using CHO/DG44 Cell into whichGMD-Targeting siRNA and FUT8-Targeting siRNA Co-Expression Vector wasIntroduced

1. Construction of GMD-Targeting siRNA and FUT8-Targeting siRNACo-Expression Vector

(1) Construction of GMD-Targeting siRNA Expression Unit in theDownstream of Human U6 Promoter

GMD-targeting siRNA expression in the downstream of human U6 promoterwas constructed using pPUR/GMDshB constructed in the item 1 of Example 1according to the following procedure (FIG. 16):

First, PUR-FW-XhoI forward primer (SEQ ID NO:78) with recognitionsequences of XhoI restriction enzyme, which binds to the upstream of thehuman U6 promoter sequence of pPUR/GMDshB, and PUR-RV-XhoI reverseprimer (SEQ ID NO:79) which binds to the downstream of the terminatorsequence of siRNA expression cassette were each designed.

PCR was carried out using DNA polymerase, KOD polymerase (manufacturedby TOYOBO) and pPUR/GMDshB as a template. Then, 20 μL of reactionsolution [KOD buffer 1 (manufactured by TOYOBO), 0.2 mmol/L dNTPs, 1mmol/L MgCl₂, 0.4 μmol/L above primers (SEQ ID NO:78 and 79), and 1U KODpolymerase] containing 10 ng of plasmid pPUR/GMDshB was prepared, andafter heating at 98° C. for 1 minute, PCR was carried out by 25 cycles,one cycle consisting of reaction at 98° C. for 15 seconds, reaction at65° C. for 5 seconds and reaction at 74° C. for 30 seconds. After thePCR, the reaction solution was subjected to agarose gel electrophoresis,and an amplified fragment (about 600 bp) containing GMD-targeting siRNAexpression cassette region in the downstream of the human U6 promoterwas recovered.

The above PCR-amplified fragment (about 600 bp) was ligated topCR-BluntII-TOPO (manufactured by Invitrogen) using Ligation High(manufactured by TOYOBO). E. coli DH5α was transformed with the reactionsolution. A plasmid DNA was isolated from the resultingkanamycin-resistant clones using QIAprep spin Mini prep Kit(manufactured by Qiagen). Hereinafter, the plasmid is referred to as“pCR/GMDshB”.

(2) Construction of GMD-Targeting siRNA and FUT8-Targeting siRNACo-Expression Vector

GMD-targeting siRNA and FUT8-targeting siRNA co-expression vector wasconstructed using plasmid pCR/GMDshB constructed in the item (1), andplasmid FT8libB/pAGE or FT8lib3/pAGE constructed in the item 4 ofExample 4 according to the following procedure (FIG. 17).

First, plasmid pCR/GMDshB was dissolved in 40 μL of NEBuffer 2(manufactured by New England Biolabs) containing 100 μg/mL BSA(manufactured by New England Biolabs), and digested with 10 units of arestriction enzyme XhoI (manufactured by New England Biolabs) at 37° C.overnight. The reaction solution was subjected to agarose gelelectrophoresis, and DNA fragment (about 600 bp) containingGMD-targeting siRNA expression cassette in the downstream of the humanU6 promoter was recovered using RECOCHIP (manufactured by TAKARA BIO).

Also, 1 μg of plasmid FT8libB/pAGE or FT8lib3/pAGE was dissolved in 40μL of NEBuffer 2 (manufactured by New England Biolabs) containing 100μg/mL BSA (manufactured by New England Biolabs), and digested with 10units of a restriction enzyme XhoI (manufactured by New England Biolabs)at 37° C. overnight. After the reaction, 15 μL of sterilized water, 4 μLof 10× alkaline phosphatase buffer, and 1 unit of alkaline phosphataseE. coli C75 (manufactured by TAKARA BIO) were added to 20 μL of thereaction solution for carrying out dephosphorylation reaction at 37° C.for 1 hour. The reaction solution was subjected to agarose gelelectrophoresis, and XhoI fragment (about 4.7 kb) derived from plasmidFT8libB/pAGE or FT8lib3/pAGE was recovered using RECOCHIP (manufacturedby TAKARA BIO).

After 5 μL of the DNA fragment (about 600 bp) containing GMD-targetingsiRNA expression cassette in the downstream of the human U6 promoter and5 μL of the XhoI fragment (about 4.7 kb) derived from plasmidFT8libB/pAGE or FT8lib3/pAGE obtained above were mixed with 10 μL ofLigation High (manufactured by TOYOBO), the mixture was allowed to reactat 16° C. overnight. E. coli DH5α (manufactured by TOYOBO) wastransformed with the reaction solution, and plasmid DNAs were isolatedfrom the resulting ampicillin-resistant clones using QIAprep spin Miniprep Kit (manufactured by Qiagen). The nucleotide sequences of eachplasmid were confirmed using DNA sequencer 377 (manufactured by PerkinElmer) and BigDye Terminator v3.0 Cycle Sequencing Kit (manufactured byApplied Biosystems) according to the manufacturer's instruction. pPURPvuII-seq-F (SEQ ID NO:61), pPUR PvuII-seq-R (SEQ ID NO:62),hu6pTsp45I/seq-F (SEQ ID NO:63), pAGE249-seqFW (SEQ ID NO:73) andpAGE249-seqRV (SEQ ID NO:74) were used as primers for sequence analysis.As a result of the sequence analysis, clones in which siRNA expressioncassette sequence was correct and GMD-targeting siRNA expressioncassette in the downstream of the human U6 promoters and FUT8-targetingsiRNA expression cassette in the downstream of the human tRNA-valpromoter were in the same direction was selected Hereinafter, plasmidsFT8libB/pAGE and FT8lib3/pAGE inserted the GMD-targeting siRNAexpression cassette in the downstream of the human hU6 promoters arereferred to as “FT8libB_GMDB/pAGE” and “FT8lib3_GMDB/pAGE”,respectively.

2. Obtaining and Culturing of Lectin-Resistant Clone CHO/DG44 byIntroducing GMD-Targeting siRNA and FUT8-Targeting siRNA Co-ExpressionVector

(1) Obtaining of Lectin-Resistant Clone by Introducing GMD-TargetingsiRNA and FUT8-Targeting siRNA Co-Expression Vector

Lectin-resistant clones were obtained by introducing FT8libB_GMDB/pAGEor FT8lib3_GMDB/pAGE, the GMD-targeting siRNA and FUT8-targeting siRNAco-expression vector constructed in the item 1 of this Example intoclone 32-05-12 according to the following procedure.

Transfection of plasmid FT8libB_GMDB/pAGE or FT8lib3_GMDB/pAGE intoclone 32-05-12 was carried out in the same manner as in the item 2(1) ofExample 1.

After the transfection, the cell suspension was suspended in a basalmedium, and inoculated into 10 dishes of 10 cm for adherent cell culture(manufactured by Falcon). After culturing under conditions of 5% CO₂ and37° C. for 24 hours, the culture supernatant was removed, and a basalmedium containing 500 μg/mL hygromycin (manufactured by WAKO)(hereinafter referred to as “Hyg500-IMDM medium”) was added, followed byculturing for further 8 days, and appeared hygromycin-resistant colonieswere counted. Furthermore, the culture supernatant was removed from someof the dishes, and LCA-IMDM medium [IMDM medium (manufactured byInvitrogen) containing 5% bovine fetal serum (manufactured byInvitrogen), 50 μg/mL gentamicin (manufactured by Nacalai Tesque), 500nmol/L MTX (manufactured by SIGMA), 1 mmol/L L-Fucose (manufactured byNacalai Tesque), and 500 μg/mL LCA (manufactured by VECTOR)] was added,followed by culturing for further 7 days. As a result, lectin-resistantclones were obtained at a high frequency by introducing either plasmidFT8libB_GMDB/pAGE or FT8lib3_GMDB/pAGE.

(2) Expansion Culture of Lectin-Resistant Clones

Lectin-resistant clones into which GMD-targeting and FUT8-targetingsiRNA co-expression vector was introduced, obtained in the item (1) wereexpansion cultured according to the following procedure.

First, the number of appeared colonies in each dish was counted. Then,lectin-resistant colonies were scraped and sucked up with a pipetteman(manufactured by GILSON) under observation with a stereoscopicmicroscope, and collected into a U-shaped-bottom 96-well plate foradherent cells (manufactured by ASAHI TECHNOGLASS). After trypsintreatment, each clone was inoculated into a flat-bottom 96-well platefor adherent cells (manufactured by Greiner), and cultured in anLCA-IMDM medium under conditions of 5% CO₂ and 37° C. overnight. Afterthe culturing, the culture supernatant was removed, and a newHyg500-IMDM medium was added thereto, followed by culturing for further9 days. After the culturing, each clone in the plate was expansioncultured in a Hyg500-IMDM medium. Hereinafter, lectin-resistant clonesobtained by introducing plasmid FT8libB_GMDB/pAGE into clone 32-05-12are referred to as “clone iBcho-H1”, “clone iBcho-H2”, “clone iBcho-H3”,“clone iBcho-H4” or “clone iBcho-H5”; those obtained by introducingplasmid FT8lib3_GMDB/pAGE into clone 32-05-12 are referred to as “clonei3cho-H1”, “clone i3cho-H2”, “clone i3cho-H3”, “clone i3cho-H4”, “clonei3cho-H5”, “clone i3cho-H6”, “clone i3cho-H7” or “clone i3cho-H8”,respectively.

3. Determination of the Amount of GMD mRNA and the Amount of FUT8 mRNAin Lectin-Resistant Clone CHO/DG44 into which GMD-Targeting siRNA andFUT8-Targeting siRNA Co-Expression Vector was Introduced

(1) Preparation of Total RNA

A total RNA was prepared from clone 32-05-12 and lectin-resistant clonesiBcho-H1, iBcho-H2, iBcho-H3, iBcho-H4, iBcho-H5, i3cho-H1, i3cho-H2,i3cho-H3, i3cho-H4, i3cho-H5, i3cho-H6, i3cho-H7 and i3cho-H8 into whichthe GMD-targeting siRNA and FUT8-targeting siRNA co-expression vectorwas introduced obtained in the item 2 of this Example according to thefollowing procedure.

Clone 32-05-12 was suspended in a basal medium, and clones iBcho-H1,iBcho-H2, iBcho-H3, iBcho-H4, iBcho-H5, i3cho-H1, i3cho-H2, i3cho-H3,i3cho-H4, i3cho-H5, i3cho-H6, i3cho-H7 and i3cho-H8 were suspended inHyg500-IMDM medium, at a density of 3×10⁵ cells/mL, and were inoculatedat 2 mL into a 6 well plate for adherent cells (manufactured byGreiner). After culturing under conditions of 5% CO₂ and 37° C. for 3days, each cell was suspended by trypsin treatment, and collected bycentrifugation at 1,000 rpm at 4° C. for 5 minutes. After the cells weresuspended in Dulbecco's PBS buffer (manufactured by Invitrogen) andre-centrifuged at 1,000 rpm at 4° C. for 5 minutes to collect cells, atotal RNA was each extracted using an RNeasy Mini Kit (manufactured byQIAGEN) according to the manufacturer's instruction. The prepared totalRNA was dissolved in 40 μL of sterilized water.

(2) Synthesis of Single-Stranded cDNA

A single-stranded cDNA was synthesized from 3 μg each of the total RNAobtained in the item (1) by reverse transcription reaction with oligo(dT) primer in a 20 μL reaction system using a SuperScriptIIIFirst-strand Synthesis System for RT-PCR (manufactured by Invitrogen)according to the manufacturer's instruction. The synthesizedsingle-stranded cDNAs were treated with RNase and the final reactionvolume was adjusted to 40 μL. In addition, each reaction solution wasdiluted 50-fold with sterilized water, and used for analysis of theamount of gene transcription described below.

(3) Determination of the Amount of GMD Gene Transcription by SYBR-PCR

The amount of mRNA transcribed from GMD and the amount of mRNAtranscribed from β-actin genes were determined according to theprocedure described in the item 2(3) of Example 5. As PCR primers,forward and reverse primers represented by SEQ ID NOs:80 and 81 wereused to amplify GMD, and forward and reverse primers represented by SEQID NOs:66 and 67 were used to amplify α-actin, respectively. Acalibration curve was obtained from measurements with the internalcontrol plasmid, and the amount of GMD mRNA and the amount of β-actinmRNA were converted into numerical terms. When the relative amount ofGMD mRNA to the amount of β-actin mRNA in clone 32-05-12 was assumed tobe 100, the comparative results of the relative amount of GMD mRNA tothe amount of β-actin mRNA are shown in FIG. 18. The amount of GMD mRNAin all the clones obtained by introducing GMD-targeting siRNA andFUT8-targeting siRNA co-expression vector were reduced to approximately10% in comparison with that in the parent clone 32-05-12.

(4) Determination of the Amount of FUT8 Gene Transcription by SYBR-PCR

The amount of mRNA transcribed from FUT8 gene and the amount of mRNAtranscribed from β-actin gene were determined according to the proceduredescribed in the item 2(4) in Example 5. As PCR primers, forward andreverse primers represented by SEQ ID NOs:75 and 76 were used to amplifyFUT8, and forward and reverse primers represented by SEQ ID NOs:66 and67 were used to amplify β-actin, respectively. A calibration curve wasobtained from measurements with the internal control plasmid, and theamount of FUT8 mRNA and the amount of β-actin mRNA were converted intonumerical terms.

When the relative amount of GMD mRNA for the amount β-actin mRNA inclone 32-05-12 was assumed to be 100, the comparative results of therelative amount of FUT8 mRNA to β-actin mRNA are shown in FIG. 19. Theamount of FUT8 mRNA in all the clones obtained by introducingGMD-targeting siRNA and FUT8-targeting siRNA co-expression vector werereduced to approximately 5% in comparison with that in the parent clone32-05-12.

4. Production and Analysis of Antibody Composition UsingLectin-Resistant Clone CHO/DG44 into which GMD-Targeting siRNA andFUT8-Targeting siRNA Co-Expression Vector was Introduced

(1) Production of Antibody Composition by Lectin-Resistant CloneCHO/DG44 into which GMD-Targeting siRNA and FUT8-Targeting siRNACo-Expression Vector was Introduced

Anti-CCR4 chimeric antibody compositions produced by clone 32-05-12 andlectin-resistant clones iBcho-H2, iBcho-H3, i3cho-H1, i3cho-H2,i3cho-H3, i3cho-H4, i3cho-H5, i3cho-H6, i3cho-H7, and i3cho-H8 intowhich GMD-targeting siRNA and FUT8-targeting siRNA co-expression vectorwas introduced obtained in the item 2 of this Example were purifiedaccording to the following procedure.

Clone 32-05-12 was suspended in basal medium, and clones iBcho-H2,iBcho-H3, i3cho-H1, i3cho-H2, i3cho-H3, i3cho-H4, i3cho-H5, i3cho-H6,i3cho-H7, and i3cho-H8 were suspended in Hyg500-IMDM medium, at adensity of 3×10⁵ cells/mL, and were inoculated at 10 mL into a T75 flaskfor adherent cells (manufactured by Greiner). After culturing underconditions of 5% CO₂ and 37° C. for 5 days, the culture supernatant wasremoved, and after washing twice with 10 mL of Dulbecco's PBS(manufactured by Invitrogen), 24 mL of EXCELL301 medium (manufactured byJRH Bioscience) was added. After culturing under conditions of 5% CO₂and 37° C. for 7 days, the each culture supernatant was recovered, andanti-CCR4 chimeric antibody compositions were purified using a MabSelectcolumn (manufactured by Amersham Bioscience) according to themanufacturer's instruction. After exchange with 10 mmol/L KH₂PO₄ bufferusing Econo-Pac 10DG (manufactured by Bio Rad), anti-CCR4 chimericantibody compositions purified from culture supernatants of variousclones were subjected to sterile filtration by using Millex GV(manufactured by MILLIPORE) of 0.22 μm pore size.

(2) Composition Analysis of Monosaccharide of Antibody Compositions

Composition analysis of monosaccharide was carried out on the anti-CCR4chimeric antibody compositions obtained in the item 4(1) of this Exampleaccording to the known method [Journal of Liquid Chromatography, 6, 1577(1983)].

Fucose(−)% calculated from the composition ratio of monosaccharide ofeach antibody are shown in Table 6. Also, when no fucose-derived peakwas detected, fucose(−)% was regarded as 100%. TABLE 6 Fucose(−) % ofanti-CCR4-chimeric antibody composition produced by each clone CloneFucose(−) % 32-05-12  4% iBcho-H2 100% iBcho-H3  99% i3cho-H1 100%i3cho-H2 100% i3cho-H3 100% i3cho-H4  98% i3cho-H5 100% i3cho-H6  98%i3cho-H7 100% i3cho-H8 100%

Fucose(−)% of antibody compositions produced by the parent clone32-05-12 before vector introduction was 4%, while fucose(−)% of antibodycompositions produced by lectin-resistant clones obtained by theintroducing GMD-targeting siRNA and FUT8-targeting siRNA co-expressionvector into the parent clone 32-05-12 were 98 to 100%, showing asignificant increase in comparison with that of the parent clone. Theseresults demonstrated that the introduction of the GMD-targeting siRNAand FUT8-targeting siRNA co-expression vector can convert CHO/DG44 cellinto cells which produce antibody compositions with low fucose content.

EXAMPLE 9

Production of Antibody Compositions Using SP2/0 Cell into which MouseGMD-Targeting siRNA and FUT8-Targeting siRNA Co-Expression Vector wasIntroduced

1. Construction of Mouse GMD-Targeting siRNA and Mouse FUT8-TargetingsiRNA Co-Expression Vector

(1) Construction of Mouse GMD-Targeting siRNA Expression Vector in theDownstream of the Human U6 Promoter

A mouse GMD gene-targeting siRNA expression vector was constructedaccording to the following procedure (FIG. 20):

First, a mouse sequence (SEQ ID NO:58) corresponding to the targetsequence (SEQ ID NO:37) of the GMD-targeting siRNA expression vector(pPUR/GMDshB) which proved effective in Chinese hamster ovary-derivedCHO/DG44 cell was selected as a target sequence. Then, a double-strandedDNA cassette was designed against the selected target sequence in thesame manner as in the item 1(3) of Example 1. Sense (hereinafterreferred to as “mGMD-B-F”) and antisense (hereinafter referred to as“mGMD-B-R”) strands of the designed synthetic oligo DNA were representedby SEQ ID NOs:82 and 83, respectively.

In addition, the nucleotide sequence of the plasmid DNA constructed byinserting double-stranded DNA, obtained by annealing the synthetic oligoDNA, into pPUR-U6term obtained in the item 1(2) of Example 1 wasdetermined in the same manner as in the item 1(4) of Example 1 using DNAsequencer 377 (manufactured by Perkin Elmer) and BigDye Terminator v3.0Cycle Sequencing Kit (manufactured by Applied Biosystems) according tothe manufacturer's instruction. pPUR PvuII-seq-F (SEQ ID NO:61) and pPURPvuII-seq-R (SEQ ID NO:62) were used as primers for sequence analysis,and the sequences of the inserted synthetic oligo DNA and ligation sitewere confirmed. Hereinafter, a plasmid inserted a double-stranded DNAobtained by annealing synthetic oligo DNA (mGMD-B-F and mGMD-B-R) arereferred to as “pPUR/GMDmB”.

(2) Obtaining of Mouse GMD-Targeting siRNA Expression Unit in theDownstream of the Human U6 Promoter

A mouse GMD-targeting siRNA expression cassette in the downstream of thehuman U6 promoter was obtained using pPUR/GMDmB constructed in the item(1) by the same method as in the item 1(1) of Example 6 (FIG. 16).

A plasmid DNA was isolated from a number of kanamycin-resistant coloniesusing QIAprep spin Mini prep Kit (manufactured by QIAGEN). Hereinafter,the plasmid is referred to as “pCR/GMDmB”.

(3) Construction of Mouse GMD-Targeting siRNA and FUT8-Targeting siRNACo-Expression Vector

A mouse GMD-targeting siRNA and FUT8-targeting siRNA co-expressionvector was constructed using plasmid pCR/GMDmB constructed in the item(2), and plasmid FT8libB/pAGE or FT8lib3/pAGE constructed in the item 4of Example 2 in the same manner as in the item 1(2) of Example 6 (FIG.17).

A plasmid DNA was isolated from the resulting ampicillin-resistantclones using QIAprep spin Mini prep Kit (manufactured by QIAGEN). Thenucleotide sequence of each of the plasmid was confirmed using DNAsequencer 377 (manufactured by Perkin Elmer) and BigDye Terminator v3.0Cycle Sequencing Kit (manufactured by Applied Biosystems) according tothe manufacturer's instruction. pPUR PvuII-seq-F (SEQ ID NO:61), pPURPvuII-seq-R (SEQ ID NO:62), hu6pTsp45I/seq-F (SEQ ID NO:63),pAGE249-seqFW (SEQ ID NO:73), and pAGE249-seqRV (SEQ ID NO:74) were usedas primers for sequence analysis.

As a result of the sequence analysis, clones in which sequences of siRNAexpression cassette were correct and mouse GMD-targeting siRNAexpression cassette in the downstream of the human U6 promoter wasinserted in the same direction as FUT8-targeting siRNA expressioncassette in the downstream of the human tRNA-val promoter were selected.Hereinafter, plasmid FT8libB/pAGE into which mouse GMD-targeting siRNAexpression cassette in the downstream of the human U6 promoter isintroduced is referred to as “FT8libB_GMDmB/pAGE” and FT8lib3/pAGEinserted mouse GMD-targeting siRNA expression cassette under the humanU6 promoters is referred to as “FT8lib3_GMDmB/pAGE”, respectively.

2. Obtaining and Culture of Lectin-Resistant Cell Line SP2/0 byIntroducing Mouse GMD-Targeting siRNA and Mouse FUT8-Targeting siRNACo-Expression Vector

Lectin-resistant clones were obtained by introducing FT8libB_GMDmB/pAGEor FT8lib3_GMDmB/pAGE, the mouse GMD-targeting and mouse FUT8-targetingsiRNA co-expression vector obtained in the item 1 of this Example, intoclone KM968 (hereinafter referred to as “clone KM968”) which is atransformant of anti-GM₂ chimeric antibody-producing mouse myeloma SP2/0cell (ATCC CRL-1581) described in Example 1 of Japanese PublishedUnexamined Patent Application No. 205694/94) according to the followingprocedure.

Transfection of various siRNA expression vector plasmids into cloneKM968 was carried out according to the following procedure byelectroporation [Cytotechnology, 3, 133 (1990)]:

First, 10 μg of each of various siRNA expression vector plasmids wasdissolved in 30 μL of NEBuffer 4 (manufactured by New England Biolabs),and digested to be linearized with 10 units of a restriction enzyme FspI(manufactured by New England Biolabs) at 37° C. overnight. After thelinearized plasmid was confirmed by agarose gel electrophoresis using apart of the reaction solution, the remaining reaction solution waspurified by phenol/chloroform extraction and ethanol precipitation, andthe recovered linearized plasmid was dissolved in 10 μL of sterilizedwater.

Also, clone KM968 was suspended in a K-PBS buffer at 1.6×10⁷ cells/mL.After 200 μL of cell suspension (3.2×10⁶) was mixed with 10 μL of theabove linearized plasmid solution, all of the cell/DNA mixture wastransferred to Gene Pulser Cuvette (Electrode interval: 2 mm)(manufactured by BIO-RAD), and transfection was carried out underconditions of 200V pulse voltage and 250 μF capacitance using a cellfusion device Gene Pulser (manufactured by BIO-RAD).

After the transfection, the cell suspension was suspended inRPMI-FBS(10)-MTX(500) medium [RPMI1640 (manufactured by Invitrogen)containing 10% fetal bovine serum (manufactured by Invitrogen) and 500nmol/L MTX (manufactured by SIGMA)], and inoculated into a T75 flask forsuspension culture (manufactured by ASAHI TECHNOGLASS). After theculture under conditions of 5% CO₂ and 37° C. for 24 hours, hygromycin(manufactured by WAKO) was added at the final concentration of 500μg/mL, and the cells were cultured for further 3 days. After theculture, the cells were collected by centrifugation at 800 rpm for 5minutes, and suspended in an RPMI-FBS(10)-MTX(500) medium containing 500μg/mL hygromycin [hereinafter referred to as“RPMI-FBS(10)-MTX(500)-Hyg(500) medium”] at a density of 0.5 to 1×10⁴viable cells/mL, and were inoculated at 100 μL into a 96-well cultureplate (manufactured by Greiner). Subsequently, the cells were culturedfor 2 weeks, while half the volume of the culture supernatant wasroutinely replaced by a new RPMI-FBS(10)-MTX(500)-Hyg(500) medium onceevery 2 to 3 days.

After the culture, the cells in each well of the 96-well culture platewere divided into two 96-well culture plates; one for a master plate,and another for a replica plate. The master and replica plates were eachcultured for 3 days in an RPMI-FBS(10)-MTX(500)-Hyg(500) medium andRPMI-FBS(10)-MTX(500)-Hyg(500) medium containing 1 mmol/L L-fucose(manufactured by Nacalai Tesque) and 500 μg/mL LCA (manufactured byVECTOR), respectively. As a result, excellent growth in the presence ofLCA was confirmed in approximately 30% of the wells of the replica platecorresponding to wells of the master plate which showed excellentgrowth.

Cells in the wells of the master plate, which corresponded to wells ofthe replica plate which showed excellent growth, were expansion culturedin an RPMI-FBS(10)-MTX(500)-Hyg(500) medium. Hereinafter, among clonesused for the expansion culture, those obtained by introducingFr8libB_GMDmB/pAGE are referred to as “clone 9681B-1”, “clone 9681B-2”,“clone 9681B-3”, “clone 9681B-4”, “clone 9681B-5”, “clone 9681B-6” and“clone 9681B-7”; those obtained by introducing FT8lib3_GMDmB/pAGE arereferred to as “clone 968i3-1”, “clone 968i3-2”, “clone 968i3-3”, “clone968i3-4”, “clone 968i3-5”, “clone 968i3-6”, “clone 968i3-7”, “clone968i3-8”, “clone 968i3-9” and “clone 968i3-10”, respectively.

3. Determination of the Amount of GMD mRNA and the Amount of FUT8 mRNAin Lectin-Resistant SP2/0 Cell into which Mouse GMD-Targeting siRNA andMouse FUT8-Targeting siRNA Co-Expression Vector was Introduced

(1) Preparation of Total RNA

A total RNA was prepared from clone KM968, and clones 9681B-1, 9681B-2,9681B-3, 9681B-4, 9681B-5, 9681B-6, 9681B-7, 968i3-1, 968i3-2, 968i3-3,968i3-4, 968i3-5, 968i3-6, 968i3-7, 968i3-8, 968i3-9 and 968i3-10 whichare lectin-resistant cell line SP2/0 into which the mouse GMD-targetingsiRNA and mouse FUT8-targeting siRNA co-expression vector was introducedobtained in item 2 of this Example according to the following procedure.

Clone KM968 was suspended in an RPMI-FBS(10)-MTX(500) medium, andlectin-resistant cell line SP2/0 into which the mouse GMD-targetingsiRNA and mouse FUT8-targeting siRNA co-expression vector was introducedwere suspended in RPMI-FBS(10)-MTX(500)-Hyg(500) medium at a density of1×10⁵ cells/mL, and inoculated into a T75 flask for suspension culture(manufactured by ASAHI TECHNOGLASS). After the culture under conditionsof 5% CO₂ and 37° C. for 3 days, the cells were counted, and 5×10⁵cells/mL each was collected. The cells were recovered by centrifugationat 800 rpm for 5 minutes. After the recovered cells were suspended inDulbecco's PBS buffer (manufactured by Invitrogen) and re-centrifuged at800 rpm for 5 minutes to recover cells, total RNA was each extractedusing an RNeasy Micro Kit (manufactured by QIAGEN) according to themanufacturer's instruction. Each prepared total RNA was dissolved in 14μL of sterilized water.

(2) Synthesis of Single-Stranded cDNA

A single-stranded cDNA was synthesized from 3 μg of each of the totalRNAs obtained in the item (1) by reverse transcription reaction witholigo (dT) primer in 20 μL reaction system using SuperScriptIIIFirst-strand Synthesis System for RT-PCR (manufactured by Invitrogen)according to the manufacturer's instruction. The synthesizedsingle-stranded cDNAs were each treated with RNase and the finalreaction volume was adjusted to 40 μL. Then, each of the reactionsolutions was diluted 50-fold with sterilized water, and used foranalysis of the amount of gene transcription described below.

(3) Determination of the Amount of GMD Gene Transcription by SYBR-PCR

The amount of mRNA transcribed from GMD gene and the amount of mRNAtranscribed from β-actin gene were determined according to the proceduredescribed in the item 3(3) of Example 8. A calibration curve wasobtained from measurements with the internal control plasmid, and theamount of GMD mRNA and the amount of β-actin mRNA were converted intonumerical terms.

When the relative amount of GMD mRNA to the amount of β-actin mRNA inclone KM968 was assumed to be 100, the comparative results of therelative mRNA amounts of GMD to the mRNA amount of β-actin are shown inFIG. 21. The amount of GMD mRNA of all the clones obtained byintroducing the mouse GMD-targeting siRNA and mouse FUT8-targeting siRNAco-expression vector were reduced to approximately 10% in comparisonwith that of the parent clone KM968.

(4) Determination of the Amount of FUT8 Gene Transcription by SYBR-PCR

The amount of mRNA transcribed from FUT8 and the amount of mRNAtranscribed from β-actin gene were determined according to the proceduredescribed in the item 3(4) of Example 8. A calibration curve wasobtained from measurements with the internal control plasmid, anddetermination of the amount of FUT8 mRNA and the amount of β-actin mRNAwere converted into numerical terms.

When the relative amount FUT8 mRNA to the amount of β-actin mRNA inclone KM968 was assumed to be 100, the comparative results of the amountof FUT8 mRNA relative to the amount β-actin mRNA are shown in FIG. 22.The amount of FUT8 mRNA of all the clones obtained by introducing themouse GMD-targeting siRNA and mouse FUT8-targeting siRNA co-expressionvector were reduced to approximately 10% in comparison with that in theparent clone KM968.

4. Production and Analysis of Antibody Composition UsingLectin-Resistant Cell Line SP2/0 into which Mouse GMD-Targeting siRNAand Mouse FUT8-Targeting siRNA Co-Expression Vector was Introduced

(1) Production of Antibody Composition by Lectin-Resistant Cell LineSP2/0 into which Mouse GMD-Targeting siRNA and Mouse FUT8-TargetingsiRNA Co-Expression Vector was Introduced

Anti-GM₂ chimeric antibody composition produced by clone KM968 andlectin-resistant clones 968i3-2 and 968i3-3 which is cell line SP2/0into which the mouse GMD-targeting siRNA and mouse FUT8-targeting siRNAco-expression vector was introduced obtained in the item 2 of thisExample, were each purified according to the following procedure.

Clone KM968 was suspended in SP-HSFM medium [Hybridoma-SFM mediumcontaining 5% UltraLow-IgG FBS (manufactured by Invitrogen) and 500nmol/L MTX (manufactured by SIGMA)], and clones 968i3-2 and 968i3-3 weresuspended in SP-HSFM medium containing 500 μg/mL hygromycin(manufactured by WAKO), at a density of 1×10⁵ cells/mL, and wereinoculated at 50 mL into a T225 flask for suspension culture(manufactured by ASAHI TECHNOGLASS). After the culture under conditionsof 5% CO₂ and 37° C. for 7 days, culture supernatants were eachrecovered, and anti-GM₂ chimeric antibody compositions were purifiedusing a MabSelect column (manufactured by Amersham Bioscience) accordingto the manufacturer's instruction. After exchange 10 mmol/L KH₂PO₄buffer using Econo-Pac 10DG (manufactured by Bio Rad), anti-CCR4chimeric antibodies purified from culture supernatants of various cloneswere subjected to sterile filtration by using Millex GV (manufactured byMILLIPORE) of 0.22 μm pore size.

(2) Composition Analysis of Monosaccharide of Antibody Compositions

Composition analysis of monosaccharide was carried out for the anti-GM₂chimeric antibody compositions obtained in the item 4(1) of this Exampleaccording to the known method [Journal of Liquid Chromatography, 6, 1577(1983)].

Fucose(−)% calculated from the composition ratio of monosaccharide ofeach antibody are shown in Table 7. TABLE 7 Fucose(−) % of anti-GM₂chimeric antibody produced by each clone Ratio of sugar chains to Clonewhich fucose is not bound (%) KM968  3% 968i3-2 64% 968i3-3 62%

Fucose(−)% of antibody compositions produced by clone KM968 was 3%,while fucose(−)% of antibody compositions produced by lectin-resistantclones 968i3-2 and 968i3-3 obtained by introducing the mouseGMD-targeting siRNA and mouse FUT8-targeting siRNA co-expression vectorinto the parent clone KM968 were approximately 60%, showing asignificant increase in comparison with that of the parent clone KM968.These results demonstrated that the introduction of the GMD-targetingsiRNA and FUT8-targeting siRNA co-expression vector can convert SP2/0cell into cells which produce antibody compositions with low fucosecontent.

EXAMPLE 10

Production of Antibody Compositions Using NS0 Cell into which MouseGMD-Targeting siRNA and Mouse FUT8-Targeting siRNA Co-Expression Vectorwas Introduced

1. Obtaining and Culturing of Cell Line NS0 into which MouseGMD-Targeting siRNA and Mouse FUT8-Targeting siRNA Co-Expression Vectorwas Introduced

Transformants were obtained by introducing FT8libB_GMDmB/pAGE orFT8lib3_GMDmB/pAGE which is a mouse GMD-targeting siRNA and mouseFUT8-targeting siRNA co-expression vector obtained in the item 1 ofExample 7 into mouse myeloma NS0 cell (RCB0213)-derived anti-CCR4chimeric antibody-producing clone NS0/2160 (hereinafter referred to as“clone NS0/2160”) which was obtained in the same manner as in item (2)of Example 1 of WO03/85118 according to the following procedure.

Transfection of various siRNA expression vector plasmids into cloneNS0/2160 was carried out according to the following procedure byelectroporation [Cytotechnology, 3, 133 (1990)].

First, 10 μg of each of various siRNA expression vector plasmids wasdissolved in 30 μL of NEBuffer 4 (manufactured by New England Biolabs),and digested to be linearized with 10 units of a restriction enzyme FspI(manufactured by New England Biolabs) at 37° C. overnight. After thelinearized plasmid was confirmed by agarose gel electrophoresis using apart of the reaction solution, the remaining reaction solution waspurified by phenol/chloroform extraction and ethanol precipitation, andthe recovered linearized plasmid was dissolved in 10 μL of sterilizedwater.

Also, clone NS0/2160 was suspended in a K-PBS buffer at 1×10⁷ cells/mL.After 200 μL of cell suspension (2×10⁶) was mixed with 10 μL of thelinearized plasmid solution, all of the cell/DNA mixture was transferredto Gene Pulser Cuvette (Electrode interval: 2 mm) (manufactured byBIO-RAD), and transfection was carried out under conditions of 200Vpulse voltage and 250 μF capacitance using a cell fusion device GenePulser (manufactured by BIO-RAD).

After the transfection, the cell suspension were suspended in anRPMI-FBS(10)-MTX(500) medium, and inoculated into a T75 flask forsuspension culture (manufactured by ASAHI TECHNOGLASS). After theculture under conditions of 5% CO₂ and 37° C. for 24 hours, hygromycin(manufactured by WAKO) was added at the final concentration of 500μg/mL, and cells were cultured for further 2 days. After the culture,cells were collected by centrifugation at 800 rpm for 5 minutes, andsuspended in RPMI-FBS(10)-MTX(500)-Hyg(500) at a density of 0.5 to 1×10⁴living cells/mL, and were inoculated at 100 μL into a 96-well cultureplate (manufactured by Greiner). Subsequently, half the volume of theculture supernatant was routinely replaced by a newRPMI-FBS(10)-MTX(500)-Hyg(500) medium once every 2 to 3 days, and thecells were cultured for 2 to 3 weeks.

After the culture, culture supernatants in each well of the 96-wellculture plates were collected, and the binding activities of anti-CCR4chimeric antibody compositions, contained in each culture supernatant,to shFcγRIIIa were evaluated by the method described in the item 3 ofExample 7. As a result of the analysis on 80 wells for each vector, forantibody compositions of the culture supernatants in 90% or more of thewells increased binding activities to shFcγRIIIa were confirmed incomparison with those of parent clone NS0/2160, demonstrating that cellsin each well was converted into cells which produce antibodycompositions with low fucose content.

Cells in 5 wells that showed increased binding activities to shFcγRIIIawere expansion cultured in the RPMI-FBS(10)-MTX(500)-Hyg(500) medium.Hereinafter, among clones used for the expansion culture, those obtainedby introducing FT8libB_GMDmB/pAGE are referred to as “cloneNS0/21601B-1”, “clone NS0/21601B-2”, “clone NS0/21601B-3”, “cloneNS0/21601B-4” and “clone NS0/21601B-5”; those by introducingFT8lib3_GMDmB/pAGE are referred to as “clone NS0/2160i3-1”, “cloneNS0/2160i3-2”, “clone NS0/2160i3-3”, “clone NS0/2160i3-4” and “cloneNS0/2160i3-5”, respectively.

2. Determination of the Amount of GMD mRNA and the Amount of FUT8 mRNAin Lectin-Resistant NS0 Cells into which Mouse GMD-Targeting siRNA andMouse FUT8-Targeting siRNA Co-Expression Vector was Introduced

(1) Preparation of Total RNA

A total RNA was prepared from clone NS0/2160, and clones NS0/21601B-1,NS0/21601B-2, NS0/21601B-3, NS0/21601B-4, NS0/21601B-5, NS0/2160i3-1,NS0/2160i3-2, NS0/2160i3-3, NS0/2160i3-4 and NS0/2160i3-5 which are cellline NS0 into which the mouse GMD-targeting siRNA and mouseFUT8-targeting siRNA co-expression vector was introduced obtained in theitem 1 of this Example according to the following procedure.

Clone NS0/2160 was suspended in an RPMI-FBS(10)-MTX(500) medium, andcell line NS0 into which a mouse GMD-targeting siRNA and mouseFUT8-targeting siRNA co-expression vector was introduced was suspendedin an RPMI-FBS(10)-MTX(500)-Hyg(500) medium, at a density of 1×10⁵cells/mL, and inoculated into a T75 flask for suspension culture(manufactured by ASAHI TECHNOGLASS). After culturing under conditions of5% CO₂ and 37° C. for 3 days, the cells were counted and 5×10⁵ cellswere each collected. The supernatant was removed by centrifugation at800 rpm for 5 minutes. After the cells were suspended in Dulbecco's PBSbuffer (manufactured by Invitrogen) and re-centrifuged at 800 rpm for 5minutes to recover cells, total RNA was each extracted using an RNeasyMicro Kit (manufactured by QIAGEN) according to the manufacturer'sinstruction. A total RNA prepared was dissolved in 14 μL of sterilizedwater.

(2) Synthesis of Single-Stranded cDNA

A single-stranded cDNA was synthesized from 3 μg each of the total RNAobtained in the item (1) by reverse transcription reaction with oligo(dT) primer in 20 μL reaction system using SuperScriptIII First-strandSynthesis System for RT-PCR (manufactured by Invitrogen) according tothe manufacturer's instruction. The synthesized single-stranded cDNAswere treated with RNase and the final reaction volume was adjusted to 40μL. Then, each of the reaction solutions was diluted 50-fold withsterilized water, and used for analysis of the amount of genetranscription described below.

(3) Determination of the Amount of GMD Gene Transcription by SYBR-PCR

The amount of mRNA transcribed from GMD gene and the amount of mRNAtranscribed from β-actin gene were determined according to the proceduredescribed in the item 3(3) of Example 8. A calibration curve wasobtained from measurements with the internal control plasmid, and theamount of GMD mRNA and the amount of β-actin mRNA were converted intonumerical terms.

When the relative amount of GMD mRNA to the amount of β-actin mRNA inclone NS0/2160 was assumed to be 100, the comparative results of therelative amount of GMD mRNA to the amount of β-actin mRNA are shown inFIG. 23. The amount of GMD mRNA in all the clones obtained byintroducing the mouse GMD-targeting siRNA and mouse FUT8-targeting siRNAco-expression vector were reduced to approximately 20% in comparisonwith that in the parent clone NS0/2160.

(4) Determination of the Amount of FUT8 Gene Transcription by SYBR-PCR

The amount of mRNA transcribed from FUT8 gene and the amount of mRNAtranscribed β-actin gene were determined according to the proceduredescribed in the item 3(4) of Example 8. A calibration curve wasobtained from measurements with the internal control plasmid, and theamount of FUT8 mRNA and the amount of β-actin mRNA were converted intonumerical terms.

When the relative amount of FUT8 mRNA to the amount of β-actin mRNA inclone NS0/2160 was assumed to be 100, the comparative results of therelative amounts of FUT8 mRNA to the amount of β-actin mRNA are shown inFIG. 24. The amount of FUT8 mRNA in all the clones obtained byintroducing the mouse GMD-targeting siRNA and mouse FUT8-targeting siRNAco-expression vector were reduced to approximately 10% in comparisonwith that in the parent clone KM968.

3. Production and Analysis of Antibody Composition Using NS0 Cell intowhich Mouse GMD-Targeting siRNA and Mouse FUT8-Targeting siRNACo-Expression Vector was Introduced

(1) Production of Antibody Composition by Antibody with Low FucoseContent Producing Cell Line NS0 into which Mouse GMD-Targeting siRNA andMouse FUT8-Targeting siRNA Co-Expression Vector was Introduced

Anti-CCR4 chimeric antibody compositions produced by clone NS0/2160, andclones NS0/21601B-3, NS0/21601B-5, NS0/2160i3-1, NS0/2160i3-4 andNS0/2160i3-5 which are cell line NS0 into which the mouse GMD-targetingsiRNA and mouse FUT8-targeting siRNA co-expression vector was introducedin the item 1 of this Example were purified according to the followingprocedure.

Clone NS0/2160 was suspended in NS-HSFM medium [Hybridoma-SFM medium(manufactured by Invitrogen) containing 0.2% bovine serum albumin(manufactured by Invitrogen) and 500 nmol/L MTX (manufactured bySIGMA)], and clones NS0/21601B-3, NS0/21601B-5, NS0/2160i3-1,NS0/2160i3-4 and NS0/2160i3-5 were suspended in NS-HSFM mediumcontaining 500 μg/mL hygromycin (manufactured by WAKO), at a density of2×10⁵ cells/mL, and were inoculated at 40 mL into a T225 flask forsuspension culture (manufactured by ASAHI TECHNOGLASS). After theculture under conditions of 5% CO₂ and 37° C. for 7 days, each culturesupernatant was recovered, and anti-CCR4 chimeric antibody compositionswere purified using a MabSelect column (manufactured by AmershamBioscience) according to the manufacturer's instruction. After exchangewith 10 mmol/L KH₂PO₄ buffer using Econo-Pac 10DG (manufactured by BioRad), anti-CCR4 chimeric antibody compositions purified from culturesupernatants of various clones were subjected to sterile filtration byusing Millex GV (manufactured by MILLIPORE) of 0.22 μm pore size.

(2) Composition Analysis of Monosaccharide of Antibody Compositions

Composition analysis of monosaccharide was carried out for the anti-CCR4chimeric antibody compositions obtained in the item 3(1) of this Exampleaccording to the known method [Journal of Liquid Chromatography, 6, 1577(1983)].

Fucose(−)% calculated from the composition ratio of monosaccharide ofeach antibody composition are shown in Table 8. TABLE 8 Fucose(−) % ofanti-CCR4 chimeric antibody composition produced by each clone CloneFucose(−) % NS0/2160 28% NS0/2160iB-3 93% NS0/2160iB-5 92% NS0/2160i3-193% NS0/2160i3-4 93% NS0/2160i3-5 92%

Fucose(−)% of the antibody compositions produced by clone NS0/2160 was28%, while fucose(−)% of the antibody compositions produced by clonesNS0/21601B-3, NS0/21601B-5, NS0/2160i3-1, NS0/2160i3-4 and NS0/2160i3-5which were obtained by introducing the mouse GMD-targeting siRNA andmouse FUT8-targeting siRNA co-expression vector into the parent cloneNS0/2160 were approximately 90%, showing a significant increase incomparison with that of the parent cell. These results demonstrated thatthe introduction of the GMD-targeting siRNA and FUT8-targeting siRNAco-expression vector can convert NS0 cells into cells which produceantibody with low fucose content.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skill in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

This application is based on Japanese application No. 2004-228928 filedon Aug. 5, 2004, Japanese application No. 2004-252682 filed on Aug. 31,2004 and Japanese application No. 2005-136410 filed on May 9, 2005, theentire contents of which are incorporated hereinto by reference. Allreferences cited herein are incorporated in their entirety.

1. A cell into which a double-stranded RNA comprising an RNA selectedfrom the following (a) or (b) and its complementary RNA are introduced:(a) an RNA comprising the nucleotide sequence represented by SEQ IDNO:37, 57 or 58; (b) an RNA consisting of a nucleotide sequence in whichone or a several nucleotide(s) is/are deleted, substituted, insertedand/or added in the nucleotide sequence represented by SEQ ID NO:37, 57or 58 and having activity of suppressing the function of an enzymecatalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose.
 2. The cell according to claim 1,wherein the enzyme catalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose is GDP-mannose 4,6-dehydratase.
 3. Thecell according to claim 2, wherein the GDP-mannose 4,6-dehydratase is aprotein encoded by a DNA selected from the group consisting of thefollowing (a) to (f): (a) a DNA comprising the nucleotide sequencerepresented by SEQ ID NO:8; (b) a DNA comprising the nucleotide sequencerepresented by SEQ ID NO:9; (c) a DNA comprising the nucleotide sequencerepresented by SEQ ID NO:10; (d) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:8 understringent conditions and encodes a protein having GDP-mannose4,6-dehydratase activity; (e) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:9 understringent conditions and encodes a protein having GDP-mannose4,6-dehydratase activity; (f) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:10 understringent conditions and encodes a protein having GDP-mannose4,6-dehydratase activity.
 4. The cell according to claim 2, wherein theGDP-mannose 4,6-dehydratase is a protein selected from the groupconsisting of the following (a) to (i): (a) a protein comprising theamino acid sequence represented by SEQ ID NO:11; (b) a proteincomprising the amino acid sequence represented by SEQ ID NO:12; (c) aprotein comprising the amino acid sequence represented by SEQ ID NO:13;(d) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:11 and having GDP-mannose4,6-dehydratase activity; (e) a protein consisting of an amino acidsequence in which one or more amino acid(s) is/are deleted, substituted,inserted and/or added in the amino acid sequence represented by SEQ IDNO:12 and having GDP-mannose 4,6-dehydratase activity; (f) a proteinconsisting of an amino acid sequence in which one or more amino acid(s)is/are deleted, substituted, inserted and/or added in the amino acidsequence represented by SEQ ID NO:13 and having GDP-mannose4,6-dehydratase activity; (g) a protein consisting of an amino acidsequence which has 80% or more homology to the amino acid sequencerepresented by SEQ ID NO:11 and having GDP-mannose 4,6-dehydrataseactivity; (h) a protein consisting of an amino acid sequence which has80% or more homology to the amino acid sequence represented by SEQ IDNO:12 and having GDP-mannose 4,6-dehydratase activity; (i) a proteinconsisting of an amino acid sequence which has 80% or more homology tothe amino acid sequence represented by SEQ ID NO:13 and havingGDP-mannose 4,6-dehydratase activity.
 5. A double-stranded RNAcomprising an RNA selected from the following (a) or (b) and itscomplementary RNA: (a) an RNA comprising the nucleotide sequencerepresented by SEQ ID NO:37, 57 or 58; (b) an RNA consisting of anucleotide sequence in which one or a several nucleotide(s) is/aredeleted, substituted, inserted and/or added in the nucleotide sequencerepresented by SEQ ID NO:37, 57 or 58 and having activity of suppressingthe function of an enzyme catalyzing a reaction which convertsGDP-mannose into GDP-4-keto,6-deoxy-G D P-mannose.
 6. A DNAcorresponding to the RNA according to claim 5 and its complementary DNA.7. A vector comprising a DNA corresponding to the RNA according to claim5.
 8. A cell into which the vector according to claim 7 is introduced.9. A cell into which an RNA capable of suppressing the function of anenzyme relating to modification of a sugar chain in which 1-position offucose is bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain, andan RNA capable of suppressing the function of an enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, or an RNAcapable of suppressing the function of a protein relating to transportof an intracellular sugar nucleotide, GDP-fucose, to the Golgi body areintroduced.
 10. A cell into which an RNA capable of suppressing thefunction of an enzyme relating to modification of a sugar chain in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in the complex type N-glycoside-linkedsugar chain, and an RNA capable of suppressing the function of an enzymerelating to synthesis of an intracellular sugar nucleotide, GDP-fucose,are introduced.
 11. The cell according to claim 9, wherein the enzymerelating to synthesis of an intracellular sugar nucleotide, GDP-fucose,is an enzyme catalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose.
 12. The cell according to claim 9,wherein the enzyme relating to modification of a sugar chain in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in the complex type N-glycoside-linkedsugar chain is α1,6-fucosyltransferase.
 13. The cell according to claim12, wherein the α1,6-fucosyltransferase is a protein encoded by a DNAselected from the group consisting of (a) to (h): (a) a DNA comprisingthe nucleotide sequence represented by SEQ ID NO:1; (b) a DNA comprisingthe nucleotide sequence represented by SEQ ID NO:2; (c) a DNA comprisingthe nucleotide sequence represented by SEQ ID NO:3; (d) a DNA comprisingthe nucleotide sequence represented by SEQ ID NO:4; (e) a DNA whichhybridizes with a DNA consisting of the nucleotide sequence representedby SEQ ID NO:1 under stringent conditions and encodes a protein havingα1,6-fucosyltransferase activity; (f) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:2 understringent conditions and encodes a protein havingα1,6-fucosyltransferase activity; (g) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:3 understringent conditions and encodes a protein havingα1,6-fucosyltransferase activity; (h) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:4 understringent conditions and encodes a protein havingα1,6-fucosyltransferase activity.
 14. The cell according to claim 12,wherein the α1,6-fucosyltransferase is a protein selected from the groupconsisting of the following (a) to (l): (a) a protein comprising theamino acid sequence represented by SEQ ID NO:5; (b) a protein comprisingthe amino acid sequence represented by SEQ ID NO:6; (c) a proteincomprising the amino acid sequence represented by SEQ ID NO:7; (d) aprotein comprising the amino acid sequence represented by SEQ ID NO:84;(e) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:5 and havingα1,6-fucosyltransferase activity; (f) a protein consisting of an aminoacid sequence in which one or more amino acid(s) is/are deleted,substituted, inserted and/or added in the amino acid sequencerepresented by SEQ ID NO:6 and having α1,6-fucosyltransferase activity;(g) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:7 and havingα1,6-fucosyltransferase activity; (h) a protein consisting of an aminoacid sequence in which one or more amino acid(s) is/are deleted,substituted, inserted and/or added in the amino acid sequencerepresented by SEQ ID NO:84 and having α1,6-fucosyltransferase activity;(i) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:5 andhaving α1,6-fucosyltransferase activity; (j) a protein consisting of anamino acid sequence which has 80% or more homology to the amino acidsequence represented by SEQ ID NO:6 and having α1,6-fucosyltransferaseactivity; (k) a protein consisting of an amino acid sequence which has80% or more homology to the amino acid sequence represented by SEQ IDNO:7 and having α1,6-fucosyltransferase activity; (l) a proteinconsisting of an amino acid sequence which has 80% or more homology tothe amino acid sequence represented by SEQ ID NO:84 and havingα1,6-fucosyltransferase activity.
 15. The cell according to claim 9,wherein the RNA capable of suppressing the function of an enzymerelating to modification of a sugar chain in which 1-position of fucoseis bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain is adouble-stranded RNA comprising an RNA selected from the group consistingof the following (a) to (d) and its complementary RNA: (a) an RNAcorresponding to a DNA comprising a nucleotide sequence represented by asequence of continued 10 to 40 bases in the nucleotide sequencerepresented by SEQ ID NO:1; (b) an RNA corresponding to a DNA comprisinga nucleotide sequence represented by a sequence of continued 10 to 40bases in the nucleotide sequence represented by SEQ ID NO:2; (c) an RNAcorresponding to a DNA comprising a nucleotide sequence represented by asequence of continued 10 to 40 bases in the nucleotide sequencerepresented by SEQ ID NO:3; (d) an RNA corresponding to a DNA comprisinga nucleotide sequence represented by a sequence of continued 10 to 40bases in the nucleotide sequence represented by SEQ ID NO:4.
 16. Thecell according to claim 9, wherein the RNA capable of suppressing thefunction of an enzyme relating to modification of a sugar chain in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in the complex type N-glycoside-linkedsugar chain is a double-stranded RNA comprising an RNA selected from thegroup consisting of the following (a) and (b) and its complementary RNA:(a) an RNA comprising the nucleotide sequence represented by any one ofSEQ ID NO:14 to 35 or 85 to 89; (b) an RNA consisting of a nucleotidesequence in which one or a several nucleotide(s) is/are deleted,substituted, inserted and/or added in the nucleotide sequencerepresented by any one of SEQ ID NO:14 to 35 or 85 to 89 and havingactivity of suppressing the function of α1,6-fucosyltransferaseactivity.
 17. The cell according to claim 11, wherein the enzymecatalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose is GDP-mannose 4,6-dehydratase.
 18. Thecell according to claim 17, wherein the GDP-mannose 4,6-dehydratase is aprotein encoded by a DNA selected from the group consisting of thefollowing (a) to (f): (a) a DNA comprising the nucleotide sequencerepresented by SEQ ID NO:8; (b) a DNA comprising the nucleotide sequencerepresented by SEQ ID NO:9; (c) a DNA comprising the nucleotide sequencerepresented by SEQ ID NO:10; (d) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:8 understringent conditions and encodes a protein having GDP-mannose4,6-dehydratase activity; (e) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:9 understringent conditions and encodes a protein having GDP-mannose4,6-dehydratase activity; (f) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:10 understringent conditions and encodes a protein having GDP-mannose4,6-dehydratase activity.
 19. The cell according to claim 17, whereinthe GDP-mannose 4,6-dehydratase is a protein selected from the groupconsisting of the following (a) to (i): (a) a protein comprising theamino acid sequence represented by SEQ ID NO:11; (b) a proteincomprising the amino acid sequence represented by SEQ ID NO:12; (c) aprotein comprising the amino acid sequence represented by SEQ ID NO:13;(d) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:11 and having GDP-mannose4,6-dehydratase activity; (e) a protein consisting of an amino acidsequence in which one or more amino acid(s) is/are deleted, substituted,inserted and/or added in the amino acid sequence represented by SEQ IDNO:12 and having GDP-mannose 4,6-dehydratase activity; (f) a proteinconsisting of an amino acid sequence in which one or more amino acid(s)is/are deleted, substituted, inserted and/or added in the amino acidsequence represented by SEQ ID NO:13 and having GDP-mannose4,6-dehydratase activity; (g) a protein consisting of an amino acidsequence which has 80% or more homology to the amino acid sequencerepresented by SEQ ID NO:11 and having GDP-mannose 4,6-dehydrataseactivity; (h) a protein consisting of an amino acid sequence which has80% or more homology to the amino acid sequence represented by SEQ IDNO:12 and having GDP-mannose 4,6-dehydratase activity; (i) a proteinconsisting of an amino acid sequence which has 80% or more homology tothe amino acid sequence represented by SEQ ID NO:13 and havingGDP-mannose 4,6-dehydratase activity.
 20. The cell according to claim11, wherein the RNA capable of suppressing the function of an enzymecatalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose is a double-stranded RNA comprising anRNA selected from the group consisting of the following (a) to (c) andits complementary RNA: (a) an RNA corresponding to a DNA consisting of anucleotide sequence represented by a sequence of continued 10 to 40bases in the nucleotide sequence represented by SEQ ID NO:8; (b) an RNAcorresponding to a DNA consisting of a nucleotide sequence representedby a sequence of continued 10 to 40 bases in the nucleotide sequencerepresented by SEQ ID NO:9; (c) an RNA corresponding to a DNA consistingof a nucleotide sequence represented by a sequence of continued 10 to 40bases in the nucleotide sequence represented by SEQ ID NO:10.
 21. Thecell according to claim 11, wherein the RNA capable of suppressing thefunction of an enzyme catalyzing a reaction which converts GDP-mannoseinto GDP-4-keto,6-deoxy-GDP-mannose is a double-stranded RNA comprisingan RNA selected from the group consisting of the following (a) and (b)and its complementary RNA: (a) an RNA comprising the nucleotide sequencerepresented by any one of SEQ ID NO:37, 57 or 58; (b) an RNA consistingof a nucleotide sequence in which one or a several nucleotide(s) is/aredeleted, substituted, inserted and/or added in the nucleotide sequencerepresented by any one of SEQ ID NO:37, 57 or 58 and having activity ofsuppressing the function of GDP-mannose 4,6-dehydratase.
 22. A DNAcomprising a DNA corresponding to an RNA capable of suppressing thefunction of an enzyme relating to modification of a sugar chain in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in the complex type N-glycoside-linkedsugar chain and its complementary DNA, and a DNA corresponding to an RNAcapable of suppressing the function of an enzyme relating to synthesisof an intracellular sugar nucleotide, GDP-fucose, and its complementaryDNA or a DNA corresponding to an RNA capable of suppressing the functionof a protein relating to transport of an intracellular sugar nucleotide,GDP-fucose, to the Golgi body and its complementary DNA.
 23. A DNAcomprising a DNA corresponding to an RNA capable of suppressing thefunction of an enzyme relating to modification of a sugar chain in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in the complex type N-glycoside-linkedsugar chain and its complementary DNA, and a DNA corresponding to an RNAcapable of suppressing the function of an enzyme relating to synthesisof an intracellular sugar nucleotide, GDP-fucose, and its complementaryDNA.
 24. The DNA according to claim 22, wherein the enzyme relating tosynthesis of an intracellular sugar nucleotide, GDP-fucose, is an enzymecatalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose.
 25. The DNA according to claim 22,wherein the enzyme relating to modification of a sugar chain in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in the complex type N-glycoside-linkedsugar chain is α1,6-fucosyltransferase.
 26. The DNA according to claim25, wherein the α1,6-fucosyltransferase is a protein encoded by a DNAselected from the group consisting of the following (a) to (h): (a) aDNA comprising the nucleotide sequence represented by SEQ ID NO:1; (b) aDNA comprising the nucleotide sequence represented by SEQ ID NO:2; (c) aDNA comprising the nucleotide sequence represented by SEQ ID NO:3; (d) aDNA comprising the nucleotide sequence represented by SEQ ID NO:4; (e) aDNA which hybridizes with a DNA consisting of the nucleotide sequencerepresented by SEQ ID NO:1 under stringent conditions and encodes aprotein having α1,6-fucosyltransferase activity; (f) a DNA whichhybridizes with a DNA consisting of the nucleotide sequence representedby SEQ ID NO:2 under stringent conditions and encodes a protein havingα1,6-fucosyltransferase activity; (g) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:3 understringent conditions and encodes a protein havingα1,6-fucosyltransferase activity; (h) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:4 understringent conditions and encodes a protein havingα1,6-fucosyltransferase activity.
 27. The DNA according to claim 25,wherein the α1,6-fucosyltransferase is a protein selected from the groupconsisting of the following (a) to (l): (a) a protein comprising theamino acid sequence represented by SEQ ID NO:5; (b) a protein comprisingthe amino acid sequence represented by SEQ ID NO:6; (c) a proteincomprising the amino acid sequence represented by SEQ ID NO:7; (d) aprotein comprising the amino acid sequence represented by SEQ ID NO:84;(e) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:5 and havingα1,6-fucosyltransferase activity; (f) a protein consisting of an aminoacid sequence in which one or more amino acid(s) is/are deleted,substituted, inserted and/or added in the amino acid sequencerepresented by SEQ ID NO:6 and having α1,6-fucosyltransferase activity;(g) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:7 and havingα1,6-fucosyltransferase activity; (h) a protein consisting of an aminoacid sequence in which one or more amino acid(s) is/are deleted,substituted, inserted and/or added in the amino acid sequencerepresented by SEQ ID NO:84 and having α1,6-fucosyltransferase activity;(i) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:5 andhaving α1,6-fucosyltransferase activity; (j) a protein consisting of anamino acid sequence which has 80% or more homology to the amino acidsequence represented by SEQ ID NO:6 and having α1,6-fucosyltransferaseactivity; (k) a protein consisting of an amino acid sequence which has80% or more homology to the amino acid sequence represented by SEQ IDNO:7 and having α1,6-fucosyltransferase activity; (l) a proteinconsisting of an amino acid sequence which has 80% or more homology tothe amino acid sequence represented by SEQ ID NO:84 and havingα1,6-fucosyltransferase activity.
 28. The DNA according to claim 22,wherein the RNA capable of suppressing the function of an enzymerelating to modification of a sugar chain in which 1-position of fucoseis bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain is anRNA selected from the group consisting of the following (a) to (d): (a)an RNA corresponding to a DNA comprising a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:1; (b) an RNA corresponding to a DNAcomprising a nucleotide sequence represented by a sequence of continued10 to 40 bases in the nucleotide sequence represented by SEQ ID NO:2;(c) an RNA corresponding to a DNA comprising a nucleotide sequencerepresented by a sequence of continued 10 to 40 bases in the nucleotidesequence represented by SEQ ID NO:3; (d) an RNA corresponding to a DNAcomprising a nucleotide sequence represented by a sequence of continued10 to 40 bases in the nucleotide sequence represented by SEQ ID NO:4.29. The DNA according to claim 22, wherein the RNA capable ofsuppressing the function of an enzyme relating to modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complextype N-glycoside-linked sugar chain is an RNA selected from the groupconsisting of the following (a) and (d): (a) an RNA comprising thenucleotide sequence represented by any one of SEQ ID NO:14 to 35 or 85to 89; (b) an RNA consisting of a nucleotide sequence in which one or aseveral nucleotide(s) is/are deleted, substituted, inserted and/or addedin the nucleotide sequence represented by any one of SEQ ID NO:14 to 35or 85 to 89 and having activity of suppressing the function ofα1,6-fucosyltransferase activity.
 30. The DNA according to claim 24,wherein the enzyme catalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose is GDP-mannose 4,6-dehydratase.
 31. TheDNA according to claim 30, wherein the GDP-mannose 4,6-dehydratase is aprotein encoded by a DNA selected from the group consisting of thefollowing (a) to (f): (a) a DNA comprising the nucleotide sequencerepresented by SEQ ID NO:8; (b) a DNA comprising the nucleotide sequencerepresented by SEQ ID NO:9; (c) a DNA comprising the nucleotide sequencerepresented by SEQ ID NO:10; (d) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:8 understringent conditions and encodes a protein having GDP-mannose4,6-dehydratase activity; (e) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:9 understringent conditions and encodes a protein having GDP-mannose4,6-dehydratase activity; (f) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:10 understringent conditions and encodes a protein having GDP-mannose4,6-dehydratase activity.
 32. The DNA according to claim 30, wherein theGDP-mannose 4,6-dehydratase is a protein selected from the groupconsisting of the following (a) to (i): (a) a protein comprising theamino acid sequence represented by SEQ ID NO:11; (b) a proteincomprising the amino acid sequence represented by SEQ ID NO:12; (c) aprotein comprising the amino acid sequence represented by SEQ ID NO:13;(d) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:11 and having GDP-mannose4,6-dehydratase activity; (e) a protein consisting of an amino acidsequence in which one or more amino acid(s) is/are deleted, substituted,inserted and/or added in the amino acid sequence represented by SEQ IDNO:12 and having GDP-mannose 4,6-dehydratase activity; (f) a proteinconsisting of an amino acid sequence in which one or more amino acid(s)is/are deleted, substituted, inserted and/or added in the amino acidsequence represented by SEQ ID NO:13 and having GDP-mannose4,6-dehydratase activity; (g) a protein consisting of an amino acidsequence which has 80% or more homology to the amino acid sequencerepresented by SEQ ID NO:11 and having GDP-mannose 4,6-dehydrataseactivity; (h) a protein consisting of an amino acid sequence which has80% or more homology to the amino acid sequence represented by SEQ IDNO:12 and having GDP-mannose 4,6-dehydratase activity; (i) a proteinconsisting of an amino acid sequence which has 80% or more homology tothe amino acid sequence represented by SEQ ID NO:13 and havingGDP-mannose 4,6-dehydratase activity.
 33. The DNA according to claim 24,wherein the RNA capable of suppressing the function of an enzymecatalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose is an RNA selected from the groupconsisting of the following (a) to (c): (a) an RNA corresponding to aDNA consisting of a nucleotide sequence represented by a sequence ofcontinued 10 to 40 bases in the nucleotide sequence represented by SEQID NO:8; (b) an RNA corresponding to a DNA consisting of a nucleotidesequence represented by a sequence of continued 10 to 40 bases in thenucleotide sequence represented by SEQ ID NO:9; (c) an RNA correspondingto a DNA consisting of a nucleotide sequence represented by a sequenceof continued 10 to 40 bases in the nucleotide sequence represented bySEQ ID NO:10.
 34. The DNA according to claim 24, wherein the RNA capableof suppressing the function of an enzyme catalyzing a reaction whichconverts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose is an RNAselected from the group consisting of the following (a) and (b): (a) anRNA comprising the nucleotide sequence represented by any one of SEQ IDNO:37, 57 or 58; (b) an RNA consisting of a nucleotide sequence in whichone or a several nucleotide(s) is/are deleted, substituted, insertedand/or added in the nucleotide sequence represented by any one of SEQ IDNO:37, 57 or 58 and having activity of suppressing the function ofGDP-mannose 4,6-dehydratase.
 35. A vector comprising the DNA accordingto claim
 22. 36. The vector according to claim 35, which comprises theDNA represented by SEQ ID NO:90 and the DNA represented by SEQ ID NO:92.37. The vector according to claim 35, which comprises the DNArepresented by SEQ ID NO:91 and the DNA represented by SEQ ID NO:92. 38.The vector according to claim 35, which comprises the DNA represented bySEQ ID NO:90 and the DNA represented by SEQ ID NO:93.
 39. The vectoraccording to claim 35, which comprises the DNA represented by SEQ IDNO:91 and the DNA represented by SEQ ID NO:93.
 40. A cell into which thevector according to claims 35 is introduced.
 41. A cell into which avector comprising a DNA corresponding to an RNA capable of suppressingthe function of an enzyme relating to modification of a sugar chain inwhich 1-position of fucose is bound to 6-position of N-acetylglucosaminein the reducing end through α-bond in the complex typeN-glycoside-linked sugar chain and its complementary DNA, and a vectorcomprising a DNA corresponding to an RNA capable of suppressing thefunction of an enzyme relating to synthesis of an intracellular sugarnucleotide, GDP-fucose, and its complementary DNA or a vector comprisinga DNA corresponding to an RNA capable of suppressing the function of aprotein relating to transport of an intracellular sugar nucleotide,GDP-fucose, to the Golgi body and its complementary DNA are introduced.42. A cell into which a vector comprising a DNA corresponding to an RNAcapable of suppressing the function of an enzyme relating tomodification of a sugar chain in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond inthe complex type N-glycoside-linked sugar chain and its complementaryDNA, and a vector comprising a DNA corresponding to an RNA capable ofsuppressing the function of an enzyme relating to synthesis of anintracellular sugar nucleotide, GDP-fucose, and its complementary DNAare introduced.
 43. The cell according to claim 41, wherein the RNAcapable of suppressing the function of an enzyme relating to synthesisof an intracellular sugar nucleotide, GDP-fucose, is an RNA capable ofsuppressing the function of an enzyme catalyzing a reaction whichconverts GDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose.
 44. The cellaccording to claim 41, wherein the enzyme relating to modification of asugar chain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in the complextype N-glycoside-linked sugar chain is α1,6-fucosyltransferase.
 45. Thecell according to claim 44, wherein the α1,6-fucosyltransferase is aprotein encoded by a DNA selected from the group consisting of (a) to(h): (a) a DNA comprising the nucleotide sequence represented by SEQ IDNO:1; (b) a DNA comprising the nucleotide sequence represented by SEQ IDNO:2; (c) a DNA comprising the nucleotide sequence represented by SEQ IDNO:3; (d) a DNA comprising the nucleotide sequence represented by SEQ IDNO:4; (e) a DNA which hybridizes with a DNA consisting of the nucleotidesequence represented by SEQ ID NO:1 under stringent conditions andencodes a protein having α1,6-fucosyltransferase activity; (f) a DNAwhich hybridizes with a DNA consisting of the nucleotide sequencerepresented by SEQ ID NO:2 under stringent conditions and encodes aprotein having α1,6-fucosyltransferase activity; (g) a DNA whichhybridizes with a DNA consisting of the nucleotide sequence representedby SEQ ID NO:3 under stringent conditions and encodes a protein havingα1,6-fucosyltransferase activity; (h) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:4 understringent conditions and encodes a protein havingα1,6-fucosyltransferase activity.
 46. The cell according to claim 44,wherein the α1,6-fucosyltransferase is a protein selected from the groupconsisting of the following (a) to (l): (a) a protein comprising theamino acid sequence represented by SEQ ID NO:5; (b) a protein comprisingthe amino acid sequence represented by SEQ ID NO:6; (c) a proteincomprising the amino acid sequence represented by SEQ ID NO:7; (d) aprotein comprising the amino acid sequence represented by SEQ ID NO:84;(e) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:5 and havingα1,6-fucosyltransferase activity; (f) a protein consisting of an aminoacid sequence in which one or more amino acid(s) is/are deleted,substituted, inserted and/or added in the amino acid sequencerepresented by SEQ ID NO:6 and having α1,6-fucosyltransferase activity;(g) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:7 and havingα1,6-fucosyltransferase activity; (h) a protein consisting of an aminoacid sequence in which one or more amino acid(s) is/are deleted,substituted, inserted and/or added in the amino acid sequencerepresented by SEQ ID NO:84 and having α1,6-fucosyltransferase activity;(i) a protein consisting of an amino acid sequence which has 80% or morehomology to the amino acid sequence represented by SEQ ID NO:5 andhaving α1,6-fucosyltransferase activity; (j) a protein consisting of anamino acid sequence which has 80% or more homology to the amino acidsequence represented by SEQ ID NO:6 and having α1,6-fucosyltransferaseactivity; (k) a protein consisting of an amino acid sequence which has80% or more homology to the amino acid sequence represented by SEQ IDNO:7 and having α1,6-fucosyltransferase activity; (l) a proteinconsisting of an amino acid sequence which has 80% or more homology tothe amino acid sequence represented by SEQ ID NO:84 and havingα1,6-fucosyltransferase activity.
 47. The cell according to claim 41,wherein the RNA capable of suppressing the function of an enzymerelating to modification of a sugar chain in which 1-position of fucoseis bound to 6-position of N-acetylglucosamine in the reducing endthrough α-bond in the complex type N-glycoside-linked sugar chain is adouble-stranded RNA comprising an RNA selected from the group consistingof the following (a) to (d) and its complementary RNA: (a) an RNAcorresponding to a DNA comprising a nucleotide sequence represented by asequence of continued 10 to 40 bases in the nucleotide sequencerepresented by SEQ ID NO:1; (b) an RNA corresponding to a DNA comprisinga nucleotide sequence represented by a sequence of continued 10 to 40bases in the nucleotide sequence represented by SEQ ID NO:2; (c) an RNAcorresponding to a DNA comprising a nucleotide sequence represented by asequence of continued 10 to 40 bases in the nucleotide sequencerepresented by SEQ ID NO:3; (d) an RNA corresponding to a DNA comprisinga nucleotide sequence represented by a sequence of continued 10 to 40bases in the nucleotide sequence represented by SEQ ID NO:4.
 48. Thecell according to claim 41, wherein the RNA capable of suppressing thefunction of an enzyme relating to modification of a sugar chain in which1-position of fucose is bound to 6-position of N-acetylglucosamine inthe reducing end through α-bond in the complex type N-glycoside-linkedsugar chain is a double-stranded RNA comprising an RNA selected from thegroup consisting of the following (a) and (b) and its complementary RNA:(a) an RNA comprising the nucleotide sequence represented by any one ofSEQ ID NO:14 to 35 or 85 to 89; (b) an RNA consisting of a nucleotidesequence in which one or more nucleotide(s) is/are deleted, substituted,inserted and/or added in the nucleotide sequence represented by any oneof SEQ ID NO:14 to 35 or 85 to 89 and having activity of suppressing thefunction of α1,6-fucosyltransferase activity.
 49. The cell according toclaim 43, wherein the enzyme catalyzing a reaction which convertsGDP-mannose into GDP-4-keto,6-deoxy-GDP-mannose is GDP-mannose4,6-dehydratase.
 50. The cell according to claim 49, wherein theGDP-mannose 4,6-dehydratase is a protein encoded by a DNA selected fromthe group consisting of the following (a) to (f): (a) a DNA comprisingthe nucleotide sequence represented by SEQ ID NO:8; (b) a DNA comprisingthe nucleotide sequence represented by SEQ ID NO:9; (c) a DNA comprisingthe nucleotide sequence represented by SEQ ID NO:10; (d) a DNA whichhybridizes with a DNA consisting of the nucleotide sequence representedby SEQ ID NO:8 under stringent conditions and encodes a protein havingGDP-mannose 4,6-dehydratase activity; (e) a DNA which hybridizes with aDNA consisting of the nucleotide sequence represented by SEQ ID NO:9under stringent conditions and encodes a protein having GDP-mannose4,6-dehydratase activity; (f) a DNA which hybridizes with a DNAconsisting of the nucleotide sequence represented by SEQ ID NO:10 understringent conditions and encodes a protein having GDP-mannose4,6-dehydratase activity.
 51. The cell according to claim 49, whereinthe GDP-mannose 4,6-dehydratase is a protein selected from the groupconsisting of the following (a) to (i): (a) a protein comprising theamino acid sequence represented by SEQ ID NO:11; (b) a proteincomprising the amino acid sequence represented by SEQ ID NO:12; (c) aprotein comprising the amino acid sequence represented by SEQ ID NO:13;(d) a protein consisting of an amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted, inserted and/or added in theamino acid sequence represented by SEQ ID NO:11 and having GDP-mannose4,6-dehydratase activity; (e) a protein consisting of an amino acidsequence in which one or more amino acid(s) is/are deleted, substituted,inserted and/or added in the amino acid sequence represented by SEQ IDNO:12 and having GDP-mannose 4,6-dehydratase activity; (f) a proteinconsisting of an amino acid sequence in which one or more amino acid(s)is/are deleted, substituted, inserted and/or added in the amino acidsequence represented by SEQ ID NO:13 and having GDP-mannose4,6-dehydratase activity; (g) a protein consisting of an amino acidsequence which has 80% or more homology to the amino acid sequencerepresented by SEQ ID NO:11 and having GDP-mannose 4,6-dehydrataseactivity; (h) a protein consisting of an amino acid sequence which has80% or more homology to the amino acid sequence represented by SEQ IDNO:12 and having GDP-mannose 4,6-dehydratase activity; (i) a proteinconsisting of an amino acid sequence which has 80% or more homology tothe amino acid sequence represented by SEQ ID NO:13 and havingGDP-mannose 4,6-dehydratase activity.
 52. The cell according to claim43, wherein the RNA capable of suppressing the function of an enzymecatalyzing a reaction which converts GDP-mannose intoGDP-4-keto,6-deoxy-GDP-mannose is a double-stranded RNA comprising anRNA selected from the group consisting of the following (a) to (c) andits complementary RNA: (a) an RNA corresponding to a DNA consisting of anucleotide sequence represented by a sequence of continued 10 to 40bases in the nucleotide sequence represented by SEQ ID NO:8; (b) an RNAcorresponding to a DNA consisting of a nucleotide sequence representedby a sequence of continued 10 to 40 bases in the nucleotide sequencerepresented by SEQ ID NO:9; (c) an RNA corresponding to a DNA consistingof a nucleotide sequence represented by a sequence of continued 10 to 40bases in the nucleotide sequence represented by SEQ ID NO:10.
 53. Thecell according to claims 43, wherein the RNA capable of suppressing thefunction of an enzyme catalyzing a reaction which converts GDP-mannoseinto GDP-4-keto,6-deoxy-GDP-mannose is a double-stranded RNA comprisingan RNA selected from the group consisting of the following (a) and (b)and its complementary RNA: (a) an RNA comprising the nucleotide sequencerepresented by any one of SEQ ID NO:37, 57 or 58; (b) an RNA consistingof a nucleotide sequence in which one or a several nucleotide(s) is/aredeleted, substituted, inserted and/or added in the nucleotide sequencerepresented by any one of SEQ ID NO:37, 57 or 58 and having activity ofsuppressing the function of GDP-mannose 4,6-dehydratase.
 54. The cellaccording to claim 1, which is resistant to a lectin which recognizes asugar chain structure in which 1-position of fucose is bound to6-position of N-acetylglucosamine in the reducing end through α-bond ina complex type N-glycoside-linked sugar chain.
 55. The cell according toclaim 54, wherein the lectin is selected from the group consisting ofthe following (a) to (d): (a) a Lens culinaris agglutinin LCA (lentilagglutinin derived from Lens culinaris); (b) a Pisum sativum agglutininPSA (pea lectin derived from Pisum sativum); (c) a Vicia faba agglutininVFA (agglutinin derived from Vicia faba); (d) an Aleuria aurantia lectinAAL (lectin derived from Aleuria aurantia).
 56. The cell according toclaim 1, which is a cell selected from the group consisting of an yeast,an animal cell, an insect cell and a plant cell.
 57. The cell accordingto claim 56, wherein the animal cell is selected from the groupconsisting of the following (a) to (k): (a) a CHO cell derived from aChinese hamster ovary tissue; (b) a rat myeloma cell lineYB2/3HL.P2.G11.16Ag.20 cell; (c) a mouse myeloma cell line NS0 cell; (d)a mouse myeloma cell line SP2/0-Ag14 cell; (e) a BHK cell derived from aSyrian hamster kidney tissue; (f) a hybridoma cell which produces anantibody; (g) a human leukemic cell line Namalwa cell; (h) a humanleukemic cell line NM-F9 cell; (i) a human embryonic retinal cell linePER.C6 cell; (j) an embryonic stem cell; (k) a fertilized egg cell. 58.The cell according to claim 1, which comprises a gene encoding aglycoprotein.
 59. The cell according to claim 58, wherein theglycoprotein is an antibody molecule.
 60. The cell according to claim59, wherein the antibody molecule is selected from the group consistingof the following (a) to (d): (a) a human antibody; (b) a humanizedantibody; (c) an antibody fragment comprising the Fc region of (a) or(b); (d) a fusion protein comprising the Fc region of (a) or (b). 61.The cell according to claim 59, wherein the antibody molecule belongs toan IgG class.
 62. A process for producing a glycoprotein composition,which comprises using the cell according to claim
 58. 63. A process forproducing a glycoprotein composition, which comprises culturing the cellaccording to claim 58 in a medium to form and accumulate theglycoprotein composition in the culture; and recovering and purifyingthe glycoprotein composition from the culture.
 64. A process forproducing an antibody composition, which comprises using the cellaccording to claim
 59. 65. A process for producing an antibodycomposition, which comprises culturing the cell according to claim 59 ina medium to form and accumulate the antibody composition in the culture;and recovering and purifying the antibody composition from the culture.66. The process according to claim 64, wherein the antibody compositionis an antibody composition having a higher antibody-dependentcell-mediated cytotoxic activity than an antibody composition producedby its parent cell.
 67. The process according to claim 66, wherein theantibody composition having a higher antibody-dependent cell-mediatedcytotoxic activity has a higher ratio of a sugar chain in which fucoseis not bound to N-acetylglucosamine in the reducing end in the sugarchain among total complex type N-glycoside-linked sugar chains bound tothe Fc region in the antibody composition than an antibody compositionproduced by its parent cell.
 68. The process according to claim 67,wherein the sugar chain in which fucose is not bound is a sugar chain inwhich 1-position of the fucose is not bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in a complex typeN-glycoside-linked sugar chain.
 69. A glycoprotein composition producedby the process according to claim
 62. 70. An antibody compositionproduced by the process according to claim
 64. 71. A medicamentcomprising the composition according to claim 69 as an activeingredient.